WO2009040508A2 - Methods of detection and enrichment of prpsc by immunoprecipitation and elisa - Google Patents

Abstract

The invention relates to a method of extracting or enriching PrP sc from a sample, said method comprising: (i) contacting said sample with a reagent which selectively binds PrPsc (ii) incubating said reagent with said sample (iii) separating or concentrating said reagent from said sample wherein said reagent comprises an ICSM33 antibody or a fragment or fusion thereof. The invention also relates to methods of detection of PrPsc, and to buffers and materials for use in same.

Description

METHODS FIELD OF THE INVENTION

The invention relates to the manipulation and detection of PrPsc. In particular, the invention relates to the enrichment of and detection of PrPsc from samples such as blood.

BACKGROUND TO THE INVENTION

The prion diseases, which include Creutzfeldt- Jakob disease (CJD) and kuru in humans, and scrapie and bovine spongiform encephalopathy (BSE) in ruminants, are a group of fatal, transmissible, neurodegenerative conditions. The human prion diseases are unique in that they may have a sporadic, inherited or acquired aetiology ^l and it is now known that BSE prions have infected humans ^2"5. The central feature of prion diseases is the post-translational conversion of the normal cellular prion protein (PrP^c), a host-encoded, glycosylphosphotidylinositol (GPI)- anchored sialoglycoprotein ⁶, into an abnormal isoform termed PrP^Sc.

The conformation of PrP^c was first established by NMR measurements made on the recombinant mouse protein ⁷. Since then, NMR measurements on PrP from numerous mammalian species, including human PrP ^" , show that they have essentially the same conformation with predominantly α-helical structure. To date, however, there has been no detailed structural characterisation of PrP^Sc, although both circular dichroism and Fourier transform infrared spectroscopic methods have shown PrP^Sc is composed of predominantly β-sheet structure ^11>12. There are no known covalent post-translational modifications .between PrP^c and PrP^{Sc 13} the difference between the two being purely conformational leading to physico- chemical differences that can be exploited for differentiation. In particular, PrP^c can be completely digested by the serine protease, proteinase K (PK), whereas only 90-100 amino acids are removed from the N-terminus of PrP^Sc by PK¹⁴'¹⁵, depending upon prion strain type, leaving a large protease-resistant C-terminal fragment which can be detected by immunoassays and is recognised as a key marker of CJD prion infection. Although the quantities of PrP^Sc deposited in neural tissues is sufficient for detection by western blotting, the levels in . peripheral tissues are significantly lower^16"18.

Disease-associated prion protein, PrP^Sc, is a reliable marker of prion disease and its detection has become a standard method for the diagnosis of vCJD. A major limitation of this strategy is the amino acid sequence identity of the normal cellular form of the prion protein, PrP^c, and PrP^Sc resulting in cross-reactivity in immunoassays. The low abundance of PrP^Sc against a background of ubiquitously expressed PrP^c has always required depletion of PrP⁰, usually by proteolytic digestion of the diagnostic specimen, which can create problems such as loss of the protease sensitive component of PrPsc.

The development of diagnostic tests that could be applied to peripheral tissues accessible without surgery - and ideally blood - have proved exigent due to the both the low levels of PrP^Sc and the large excess of PrP^c. Both normal and disease- associated isoforms of PrP are recognised by anti-PrP antibodies and prior art diagnostic tests depend upon protease pre-treatment of tissue samples to degrade background PrP^c allowing specific detection of PrP^Sc. However, such protease treatment may be associated with a concomitant loss of a significant proportion of PrP^Sc. There is also increasing evidence for the existence of significant quantities disease-associated isoforms of PrP which are sensitive to PK digestion ^l9"26.

Significant effort has. been directed towards the production of an antibody specific for PrP^Sc as such an antibody would obviate the problems associated with the ubiquitous expression of PrP^c. These efforts have been based upon the presumption that PrP may contain unique epitopes not present in PrP and that antibodies with conformation-dependent epitopes could be identified. To-date however, only one monoclonal antibody (niAb) 15B3 ²⁷ and plasminogen ²⁸'²⁹, have been reported to specifically bind PrP^Sc, albeit with apparently low affinity. However, neither of these developments has led to any practical advance in prion diagnostics. Recently antibodies raised against a polypeptide containing a Tyr-Tyr- Arg motif were reported to specifically bind PrP^{Sc 30}. However like 15B3, these mAbs are exclusively IgM and no utility in sensitive diagnostic assays has been established.

As well as the conformational differences that exist between PrP⁰ and PrP^Sc the highly aggregated nature of disease-associated PrP has been exploited to allow selective immunoprecipitation of PrP^Sc31. However, in the latter study recognition of PrP^Sc from human brain homogenates required PK cleavage to obtain efficient precipitation, thereby negating the potential benefit of the specific binding properties. Despite concerted efforts, to date no effective diagnostic method has yet been developed based upon an antibody with selectivity for PrP^Sc, which is a problem in the art.

Detection of disease-associated, abnormal forms of the prion protein such as PrP^Sc is the most widely used and specific criterion for the diagnosis of prion disease in humans and animals ' . However, the high specificity associated with PrP detection has always been counterbalanced by a limit on the sensitivity of detection. This limit is a product not only of the absolute levels of PrP^Sc present in a particular diagnostic specimen but also the ratio of PrP^c to PrP^Sc in that sample. Most prior art or classical immunisation strategies designed to raise anti-PrP antibodies have only yielded antibodies that recognise native PrP^c, not native PrP^Sc, and their use has been limited to applications where PrP is detected in a denatured state. Detection of denatured PrP by western blotting or ELISA results in cross-reactivity between all forms of PrP with the monoclonal antibodies currently used in the art in the diagnosis of prion disease. Their use therefore requires pre-treatment of samples before analysis, typically proteolytic digestion with PK which selectively degrades PrP . However it is now accepted that prion infection is associated with deposition of protease-sensitive abnormal isoforms of PrP ^19>20'²²-²⁶. indeed, it now appears that the majority of disease related PrP maybe destroyed by PK under conditions that are typically employed to detect prototypical PrP^{Sc 23}'²⁶. The highest level of discrimination between PrP^c and PrP^Sc that can be achieved by PK digestion is approximately 1000 fold and it is this ratio, a reflection of the relative sensitivity to PK digestion of PrP^c and PrP^Sc, that limits the sensitivity of prior art diagnostic assays for PrP^Sc. It is now apparent that the development of new diagnostic methods that do not rely on protease digestion are required.

There have been previous reports of PrP^Sc-specific antibodies, including the IgM antibody 15B3 raised against recombinant bovine PrP, which has been claimed to recognise three linear epitopes of the PrP primary sequence which are presented as a conformational epitope in PrP^Sc but not PrP^{c 27}. However, 15B3 appears to be ineffective in passive immunisation experiments³⁹ and has yet to be shown to have reliable diagnostic value.

Antibodies have also been raised against a short peptide motif, tyrosine-tyrosine- arginine (YYR) ³⁰ based upon the hypothesis that this motif is normally buried in PrP^c but is exposed in PrP^Sc. However, these antibodies have no demonstrable specific recognition of PrP and do not cross-react with the peptide used to generate them. A detailed study of the interactions between different PrP isoforms and a wide range of antibodies has provided a plausible explanation for previous observations that some antibodies can be demonstrated to selectively precipitate PrP^{Sc 31}. These authors describe immunoprecipitation reactions from normal and diseased human and ovine brain homogenates with a panel of both anti-PrP and unrelated antibodies. Several antibodies were identified that could immunoprecipiate PrP^Sc with varying degrees of selectivity over PrP^c including antibodies that were raised against unrelated, non-PrP immunogens. Morel et al hypothesise that the aggregated and hydrophobic nature of PrP^Sc leads to non-specific but selective binding to a range of substrates including BSA-conjugated magnetic beads, hi all instances where selectivity could be demonstrated with an intact IgG or IgM molecule the equivalent Fab fragment was unable to immunoprecipitate PrP^Sc. Although some of the antibodies described by Morel et al achieved good levels of discrimination between PrP isoforms, pre-treatment of the infected human brain homogenates with PK was required in order to obtain the maximal level of immunoprecipitation.

The invention seeks to overcome problems associated with the prior art.

SUMMARY OF THE INVENTION

Pre-symptomatic detection of prion disease is a problem in the art. hi particular, detection of prions from readily available biological samples such as blood has been extremely difficult. Indeed, prior art enrichment techniques such as proteinase K treatment also have a destructive effect on a subset of the PrPsc which it is desired to detect. Therefore, although prior art based techniques can increase the PrPsc signal relative to the PrP signal, overall they lead to a partial destruction or reduction of the PrPsc signal itself.

The present inventors have provided a reagent which permits selective isolation of PrPsc. This has not been possible in the prior art. This selective reagent provides a novel technique for the non-destructive enrichment or capture of PrPsc. Therefore, the majority of or even all of the PrPsc signal is preserved in assays according to the invention. Moreover,, this enrichment is itself advantageous in embodiments involving amplification, since by enriching a greater input into the amplification system is achieved and therefore the amplification can be shortened or abbreviated. In addition to the specific reagents described herein, the inventors have identified optimised conditions for binding PrPsc and for conducting the assays. These advantageously permit harvesting of PrPsc from complex and difficult biological samples such as blood, as well as a very sensitive and specific detection of PrPsc. Thus, the invention is based upon these surprising findings.

In one aspect the invention provides a method of extracting or enriching PrPsc from a sample, said method comprising: (i) , contacting said sample with a reagent which selectively binds PrPsc

(ii) incubating said reagent with said sample (iii) separating or concentrating said reagent from said sample wherein said reagent comprises an ICSM33 antibody or a fragment or fusion thereof.

In another aspect, the invention relates to a method of extracting or enriching PrPsc from a sample as described above further comprising dilution of said sample with buffer comprising detergent in an amount effective for cell membrane disruption and comprising protein in an amount effective to ameliorate non-specific binding ofreagents.

In another aspect, the invention relates to a method as described above wherein separating said PrP bound reagent comprises capturing said antibody or fragment or fusion thereof by attachment to a solid phase substrate.

In another aspect, the invention relates to a method for amplification of PrPsc, said method comprising extracting or enriching PrPsc as described above, and then amplifying said extracted or enriched PrPsc. Suitably amplification is by protein misfolding cyclic amplification (PMCA).

In another aspect, the invention relates to a method for detection of PrPsc in a sample, said method comprising extracting or enriching PrPsc as described above, optionally amplifying said PrPsc, and detecting said PrPsc by ELISA.

In another aspect, the invention relates to a method for detection of PrPsc in a sample, said method comprising detecting said PrPsc by ELISA, wherein detection comprises dilution with buffer comprising detergent in an amount effective for cell membrane disruption and protein in an amount effective to ameliorate non-specific binding ofreagents to the ELISA substrate.

In another aspect, the invention relates to a method as described above wherein said buffer comprises Tris.Cl pH 8.4, sodium lauroylsarcosine, Triton X-100, and bovine serum albumin (BSA) in the ratio 1 : 3.17 : 3.17 : 3.17. Suitably said sample is diluted with said buffer to give a final concentration of 5OmM Tris.Cl pH 8.4, 2.5% w/v sodium lauroylsarcosine, 2.5% v/v Triton X-100, and 2.5% w/v bovine serum albumin (BSA).

In another aspect, the invention relates to a method as described above wherein said ELISA detection is detection via binding to an antibody selected from ICSMlO, ICSMl 8, ICSM33 or ICSM35 antibody or a fragment or fusion thereof.

Suitably said ELISA detection is detection via binding to ICSMlO antibody or a fragment or fusion thereof.

In another aspect, the invention relates to use of ICSM33 antibody or a fragment or fusion thereof in the selective immunoprecipitation of PrPsc.

In another aspect, the invention relates to use of ICSM33 antibody or a fragment or fusion thereof in the specific capture of PrPsc.

In another aspect, the invention relates to use of ICSM33 antibody or a fragment or fusion thereof in the ELISA selective detection of PrPsc.

In another aspect, the invention relates to a buffer for the manipulation of PrP, said buffer comprising Tris.Cl pH 8.4, sodium lauroylsarcosine, Triton X-100, and bovine serum albumin (BSA) in the ratio 1 : 3.17 : 3.17 : 3.17.

In another aspect, the invention relates to a buffer as described above wherein said buffer has a concentration of 5OmM Tris.Cl pH 8.4, 2.5% w/v sodium lauroylsarcosine, 2.5% v/v Triton X-100, and 2.5% w/v bovine serum albumin (BSA). This is particularly advantageous for provision of optimal conditions for selective binding of PrPsc.

In another aspect, the invention relates to use of a buffer as described above in the immunoprecipitation of PrPsc. In another aspect, the invention relates to use of a buffer as described above in the ELISA detection of PrPsc.

In another aspect, the invention relates to a method as described above wherein said ICSM33 antibody comprises IgG₂b mouse immunoglobulin raised against soluble beta-PrP⁹¹-²³¹.

In another aspect, the invention relates to a method as described above wherein said ICSM33 antibody comprises amino acid sequence or comprises CDR amino acid sequence encoded by at least one ICSM33 nucleotide sequence selected from the sequence listing, or a homologue thereof.

In another aspect, the invention relates to a method or use as described above wherein said sample comprises blood, or said PrPsc is present in a sample comprising blood. This is particularly advantageous since prior art techniques are prone to failure in application to blood.

DETAILED DESCRIPTION OF THE INVENTION

Our observations support the hypothesis that it is the aggregated nature of PrP^Sc that allows it to be selectively isolated from PrP^c by immunoprecipitation involving multivalent interactions. However, in contrast to previously described antibodies, the recognition of PrP by ICSM33 is antigen-specific and is mediated via a linear epitope encompassing residues 93-107. The intact IgG molecule can bind to both PrP^c and PrP^Sc from all of the species tested in native conditions. Recognition of human, bovine and ovine PrP is of low affinity and the inclusion of any wash step as part of an immunoprecipiation or western blotting protocol is sufficient to disrupt binding, whereas binding to PrP^Sc is of significantly higher affinity and can withstand repeated, prolonged wash steps allowing specific detection of native PrP^Scby immunoprecipitation.

The strength of the binding affinity of ICSM33 for PrP appears to be dependent upon PrP residue 97 which is required to be asparagine in order for the antibody to bind tightly. In species that express asparagine at residue 97 (hamster and mouse) the antibody affinity for PrP^c is high and no selective immunoprecipitation is observed. In addition ICSM33 can be used as primary antibody in western blotting procedures to detect denatured PrP from these species. The affinity of ICSM33 for PrP^c appears to be delicately poised and residues other than aspargine at position 97 disrupt binding. However, although the affinity for PrP^c is substantially lowered, it is not abolished and recognition of PrP^Sc is unaffected due to multivalent interactions with the IgG-protein G matrix. We have been able to exploit this unique property in an immunoprecipitation method that can be used to detect vCJD infection in crude brain homogenates without the need for proteolytic pre-processing. In this study the method was found to be 100% specific and 100% sensitive with six infected brain homogenates being correctly identified from a set of twenty samples analysed in a blinded manner. The discrimination between PrP^c and PrP was absolute with no detectable background of PrP .

It is known that there maybe undetectable or barely detectable levels of PK- resistant PrP in certain inherited prion diseases ¹^⁰'⁴¹. We have also applied the method of the invention to samples obtained from two cases of inherited prion disease where, despite the presence of abnormal PrP deposition in immunohistological sections, conventional PK-based western blotting was unable to detect the presence of abnormal PrP. Immunoprecipitation with ICSM33 revealed the presence disease-associated PrP and thus can. be demonstrated to be more sensitive than conventional methods.

Thus the ability to specifically detect PrP^Sc without the limitations of PK digestion allows far greater levels of discrimination between the normal and disease- associated isoforms of PrP which are required for sensitive diagnosis from peripheral tissues and fluids where the ratio of PrP^Sc to PrP^c is much lower than in the central nervous system and lymphoreticular tissues.

The term "enrichment" refers to the production of a greater concentration of the target molecule, typically PrPsc. Extraction refers to the removal of the substance of interest from the sample. Extraction may therefore involve the complete removal of the sample, such as by washing away of the sample whilst retaining the captured PrPsc and thereby extracting it. Enrichment differs, from extraction in that residual sample may still remain in the case of enrichment. Enrichment has a meaning of concentration (increasing concentration). For example, it would be considered that PrPsc had been enriched if its concentration was increased in the sample. However, extraction has a meaning of isolation, in other words that the PrPsc should be isolated from at least one or more elements of the starting sample. Thus, extraction implies separation of the PrPsc from one or more components of the sample. Enrichment of PrPsc does not imply total isolation, but rather implies an increase in concentration of PrPsc, whether or not one or more components of the original sample persist after the enrichment.

The sample may be any suitable sample of interest. For example, the sample may be organ homogenate, biopsy homogenate, or any biological sample such as a bodily fluid e.g. blood. Furthermore, the sample may comprise a whole or part of an unhomogenised tissue or organ. Suitably, the sample is blood. It is an advantage of the invention that immunoprecipitation of PrPsc, particularly selective immunoprecipitation of PrPsc, may be undertaken directly on blood. It is an advantage of the invention that ELISA detection of PrPsc, particularly selective

ELISA detection of PrPsc, may be conducted directly on blood. By "directly on blood" is meant either blood (whole blood) or a diluted sample of blood. It is an advantage of the invention that extraction of PrPsc from blood prior to ELISA is optional.

When the sample of the invention is blood, suitably human blood, suitably the methods of the invention do not involve collection of the blood but rather are suitably in vitro methods carried out on blood which has previously been collected.

Suitably each study or method is used to assay approximately lOmls of blood (or enriched material equivalent to 10ml blood). This enables a robust distinction between positive and negative results to be achieved. This also enables good sensitivity to be attained.

Amelioration of non-specific binding of reagents has its normal meaning in the art. Typically, this means control of or reduction of background binding (i.e. non specific binding) of the elements of the assay to the solid phase substrates used.

Clearly, total elimination of non-specific binding may not be attainable for a given set of conditions. However, the amelioration of non-specific binding is optimised in order to provide a workable signal to noise ratio. In other words, so long as background or non-specific binding is reduced to an acceptable level which does not perturb the conduct of the assay, then that binding has been ameliorated.

A solid phase substrate may be any suitable substrate. For example, a solid phase substrate may comprise the surface of an ELISA well. Alternatively, the solid phase substrate may comprise a bead for antibody capture such as a protein A coated magnetic bead as is well known in the art.

ANTIBODIES

The antibodies used herein are each commercially available. For example, antibodies are typically obtained from a commercial source such as from D-Gen Ltd. In particular, antibodies ICSMlO, ICSM18, ICSM33, and ICSM35 are as supplied by D-Gen Ltd (UK).

A designation of "B" following an antibody indicates that it is biotinylated. For example, ICSMl OB means ICSMl 0 biotinylated antibody.

ICSMlO is capable of recognising native PrPsc. ICSMlO predominantly binds monoglycosylated or unglycosylated prion protein (PrP). ICSMlO has low or negligible affinity for diglycosylated PrP.

ICSMlO may be used to capture/enrich the prion material. ICSMlO also has the advantage of binding plate/plastic material. For example, ICSMlO may be used as the capture/enrichment agent which is advantageous since ICSMlO binds plastic matrix such as ELISA plates/plastic matrix/plastic substrate. Thus in a preferred embodiment the method of the invention involved an ICSMlO capture/enrichment step followed by an amplification step optionally followed by ELISA detection.

It is a key feature of the present invention that ICSM33 is used as a selective antibody, selective for PrPsc. In particular, it is disclosed herein for the first time that ICSM33 is selective for PrPsc over PrPc. It is this selectivity which provides numerous advantages of the invention. Selectivity for PrPsc means that the antibody binds PrPsc and does not bind, or binds at only a negligible or de minimis level, to PrPc, thereby providing selectivity or specificity for PrPsc.

Use of a PrPsc selective reagent, such as ICSM33, advantageously allows the avoidance or omission of a proteinase K digestion step. A proteinase K digestion step is usually employed in the art in order to enrich PrPsc. Although proteinase K exhibits a small amount of activity in degrading PrPsc, it exhibits a far greater activity in degrading PrPc. In addition, there is a sub population of PrPsc which is recalcitrant or resistant to proteinase K activity. Therefore, by proteinase K treatment of a sample of prion protein, PrPsc is dramatically enriched and PrPc is dramatically reduced or degraded. By using selective PrPsc binding reagents as taught in the present invention, a protease degradation step such as a step involving proteinase K, can be advantageously omitted. This is because enrichment is preferably brought about by binding to the PrPsc selective reagent. Suitably the PrPsc selective reagent is ICSM33 antibody or a fragment or derivative thereof. , .

The PrPsc selective reagent of the present invention may comprise one or more antibodies or antibody fragments capable of binding prion protein PrPsc, mimetics thereof or small molecule(s) capable of binding prion protein PrPsc or combinations thereof. Preferably the PrPsc selective reagent of the invention is an antibody or fragment thereof, preferably a monoclonal antibody or fragment thereof. Preferably the agent of the invention comprises an antibody or antibody fragment capable of selectively binding prion protein PrPsc, such as ICSM33 antibody or a fragment thereof.

Preferably the antibody comprises at least the CDRs of one or more antibodies shown in the sequence listing. Preferably the antibody is ICSM33 or a fragment or fusion thereof.

Advantageously when the agent is . an antibody, said antibody is a humanised antibody. Humanisation of antibodies is well known in the art and can be easily accomplished by the skilled worker. For example, ICSM33 or other antibody mentioned herein may be humanised with reference to the sequences encoding the CDRs presented herein. In this regard,

SEQ ID NO 1 corresponds to ICSM35VH;

SEQ ID NO: 2 corresponds to ICSM35VK;

SEQ ID NO: 3 corresponds to ICSMl 8VH;

SEQ ID NO: 4 corresponds to ICSMl 81c.

The sequences of ΪCSM33 are as follows:

ICSM33 VH sequence:

MEWT WVF LF LLS VTEG VH S Q VQLQ QS GP EL VKP GAS VKISCKASGYAFSNSWMNWVKQRPGKGLEWIGRIYL GDGDTNYNGKFKGKATLTADKSSNTAYMQLSSLTSE DSAVYFCARAPLRYPYFDYWGQGTTLTVSSA

ICSM33 VK sequence:

MVSTAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGD RVTISCRASQDISNYLNWYQQKPDGTVKLLISYTSRL HSGVPSRFSGSGSGTDYFLTISNLEQEDIATYFCQQGN TLPPTFGGGTKLEIKRADAAPTVS

Moreover, reference is made to Figure 6 which shows the CDR and other key sequences for the ICSM33 antibody.

When the antibody is or comprises ICSM33, suitably the antibody is intact such as intact IgG. The interaction with PrPsc suitably requires multivalent interactions and for this reason Fab fragments are less suitable and intact antibody offers the additional advantage of avoiding problem(s) associated with Fab fragments.

Guidance regarding humanisation may be found for example in the literature as published by Greg Winter et al., and techniques for the manipulation and production of recombinant antibodies may be found in Harlow and Lane 'Antibodies-A Laboratory Manual', Cold Spring Harbour press.

In one embodiment, the antibodies (or fragments) may advantageously be humanised by manufacture of chimaeric antibodies. hi another embodiment, the antibodies (or fragments) may advantageously be

CDR-grafted. hi another embodiment, the antibodies (or fragments) may advantageously be fully humanised to the extent that the technology permits.

The sample may be from a subject. The subject is suitably an organism, preferably a mammal, preferably a primate, preferably a human.

_|, Although ICSM33 is a poor binder of plastic/plate material, it performs well on immobilising beads. Thus, suitably ICSM33 is used in conjunction with immobilising beads.

SEQUENCE HOMOLOGY

Fragments, mutants, alleles arid other derivatives of the sequences of interest preferably retain substantial homology with said sequence. As used herein, "homology" means that the two entities share sufficient characteristics for the skilled person to determine that they are similar. Preferably, homology is used to refer to sequence identity. Thus, the derivatives of the sequences of interest preferably retain substantial sequence identity with said sequence.

Thus the present invention also relates to agents such as antibodies having CDR sequences homologous to those presented in the sequence listing, and to the uses of such antibodies and to methods involving their use as described herein.

In the context of the present invention, a homologous sequence is taken to include any sequence which is at least 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical, over at least 5, preferably 8, 10, 15, 20, 30, 40 or even more residues or bases with the sequence of interest, for example as shown in the sequence listing herein. In particular, homology should typically be considered with respect to those regions of the sequence of interest which may be known to be functionally important ie. the complementarity determining regions (CDRs) rather than non- essential neighbouring sequences such as framework regions, except of course where framework residues contribute to complementarity when such residues would be regarded as fucntionally important also. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. In some aspects of the present invention, no gap penalties are used when determining sequence identity.

Relative sequence identity may be determined by computer programs which can calculate the percentage identity between two or more sequences using any suitable algorithm for determining identity, using for example default parameters. A typical example of such a computer program is CLUSTAL (see Thompson et al., 1994 (NAR 22:4673-80) or http://www.psc.edu/general/sofrware/packages/clustal/clustal.html). Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporated herein by reference. Other computer programs used to determine identity and/or similarity between sequences include but are not limited to the GCG program package (Devereux et al 1984 Nucleic Acids Research 12:387), FASTA (Atschul et al 1990 J MoI Biol 403-410) and the GENEWORKS suite of comparison tools. Preferably, sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST. Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et si., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed₅ John Wiley & Sons, Inc.

PRION PROTEIN (PrP)

Prion protein (PrP) 'is, according to the protein only hypothesis, the primary constituent of prions. More specifically, prions are thought to consist of PrPsc which is a misfolded structured aggregate of the naturally occurring cellular constituent called PrPc. PrPsc is a functionally defined material which has the properties of proteinase K resistance and disease association. More particularly PrPsc is typically isolated ex vivo and is infectious. PrPres is used to refer to a protease resistant prion protein. PrPres may be the same as PrPsc, for example if PrPres also shown to be infectious. However, PrPres is typically generated from other material, such as recombinant material, rather than being isolated ex vivo, as an infectious agent. Nevertheless, despite there, being a precise scientific difference between PrPsc and PrPres the terms are frequently used interchangeably to refer to the proteinase K resistant PrP material.

Furthermore, it should be borne in mind that PrPsc is in fact a superset of PrP materials, and comprises disease associated PrP (sometimes called abnormal PrP) which is in fact proteinase K sensitive, as well as comprising the most proteinase K resistant fraction of disease associated PrP.

Notwithstanding the above, it is the detection or determination of the presence of PrPsc which is a main focus of the present invention.

BUFFERING

One aspect of the invention relates to new buffer systems for the analysis of PrPsc. Suitably the buffer comprises a detergent fraction and a protein fraction. Suitably the detergent fraction comprises detergent in an effective amount for the destruction of cell membranes. This has the advantage of enhancing selectivity for PrPsc over PrPc. Specifically, if membranes are not disrupted, PrPc on the cell membranes can appear as if it is an aggregated form of protein and might therefore be recognised by PrPsc specific reagents. Typically PrP aggregation permits multivalent interactions with PrPsc specific reagents such as antibodies discussed herein, and leads to a tight binding to those reagents. The clinically relevant PrPsc is indeed an aggregated form of PrP. Thus, by inclusion of detergent sufficient to disrupt cell membranes, the naturally aggregated form of PrP which is of interest, namely PrPsc, is selectively bound over PrPc which, when membranes are disrupted, is not aggregated and therefore is not bound by the PrPsc selective reagents. Most suitably the buffering is used to monomerise PrPc.

A further buffer component is a protein component. This is important in order to minimise or eliminate background signal. Some surfaces or substrates used in the analytical methods of the invention can be "sticky". This means that they have a tendency to bind proteins or polypeptides in a non-specific manner, which can lead to a background signal. Inclusion of protein in the buffer advantageously avoids this drawback. Suitably the protein is bovine serum albumin (BSA).

2x capture buffer suitably comprises 10OmM Tris.Cl pH 8.4 containing 5% w/v sodium lauroylsarcosine, 5% v/v Triton X-100, 5% w/v bovine serum albumin (BSA) and optionally 10 x Protease Inhibitor Complete cocktail (eg. as available from Roche). Thus Ix capture buffer suitably comprises 5OmM Tris.Cl pH 8.4 containing 2.5% w/v sodium lauroylsarcosine, 2.5% v/v Triton X-100, 2.5% w/v bovine serum albumin (BSA) and optionally 5 x Protease Inhibitor Complete cocktail.

Preferred buffers of the invention maybe prepared at any concentration eg. Ix, 2x, 5x, 10x etc. The buffer of the invention comprises the ingredients in fixed proportions as defined with reference to those given above, whatever the actual molarities or concentrations to which the buffer is prepared. Thus, different concentrations or stocks of buffer may be prepared and so long as they possess the fixed ratios of ingredients defined above they will be considered to fall within the scope- of the invention, ie. a buffer of the ratio derived from 0.79% Tris.Cl pH 8.4 containing 2.5% w/v sodium lauroylsarcosine, 2.5% v/v Triton X-100, 2.5% w/v bovine serum albumin (BSA) and optionally 5 x Protease Inhibitor Complete cocktail (taking the molecular weight of Tris.HCL to be 157.59); suitably the invention relates to a buffer comprising [Tris.Cl pH .8.4, sodium lauroylsarcosine,

Triton X-100, and bovine serum albumin (BSA) in the ratio 1 : 3.17 : 3.17 : 3.17] or more specifically [1 w/v Tris.Cl pH 8.4 : 3.17 w/v sodium lauroylsarcosine : 3.17 v/v Triton X-100 : 3.17 w/v bovine serum albumin (BSA)] and optionally protease inhibitors in corresponding amount.

Samples are suitably diluted to a final concentration of 1 x capture buffer. Suitably for irnmunocapture such as immunoprecipitation, said mixture will contain ICSM33 antibody to give a final volume of 200 μl and a final IgG concentration of 3μg/ml.

ELISA

Suitably the ELISA method of the invention avoids or omits the use of proteinase K.

Suitably the ELISA method of the invention uses whole blood as a substrate.

Suitably the ELISA method of the invention comprises the use of antibodies selected from the group consisting of ICSMlO, ICSMl 8, ICSM33 and ICSM35.

Suitably the ELISA method of the invention comprises the use of antibodies selected from the group consisting of ICSMlO, ICSM33 and ICSM35.

Suitably the ELISA method of the invention comprises the use of antibodies selected from the group consisting of ICSM33 and ICSMlO. Suitably the ELISA method of the invention comprises the use of antibodies selected from the group consisting of ICSMlO and ICSM35.

Most suitable antibody combinations may be selected from those presented in the examples section and accompanying figures.

PROTEASES

It is an advantage of the invention that the use of proteinase K is avoided.

Where a protease is used, aggressive proteases should be avoided. By aggressive is meant a protease having a broad spectrum of activity and/or a high level of activity. Examples of aggressive proteases whose use should be avoided are . pronase, alkase, neutrase, and proteinase K. Suitably the protease used in methods of the invention is not pronase. Suitably the protease is not alkase. Suitably the protease is not neutrase. Suitably the protease is not proteinase K.

A protease is suitably employed in the methods of the invention. An advantage of this is the elimination of false positives. Suitably the protease is a gentle acting protease. By gentle acting is meant a protease having a narrow spectrum of activity and/or a low level of activity. One example of a gentle acting protease which is particularly suitable for use in the present invention is thermolysin.

Preferably the methods of the invention involving ELISA analysis employ the use of thermolysin.

Suitably the protease treatment step is performed before dilution into ELISA buffer.

Suitably the protease treatment step is performed before any denaturation step. Suitably the protease may be inactivated or removed before ELISA analysis so as to avoid the risk of protease, degradation of the antibodies/enzymes used for detection.

DENATURATION

Denaturation is important to antigen retrieval. One advantage of the use of a denaturation step in the methods of the invention is that the greatest sensitivity can be achieved. Typically, antibody based reagents perform less well in recognition of native PrP. Therefore, it is advantageous to include a denaturation step in order to increase antibody recognition of the target PrP material.

Suitably the denaturation agent may be a detergent such as SDS. If the denaturation agent is a detergent such as SDS, it is important to dilute the samples at the appropriate point in the analysis so that a detergent does not interfere with- subsequent steps.

More suitably the denaturation agent is a guanidine based reagent. Suitably the denaturation agent may comprise four molar guanidium hydrochloride.

Suitably the denaturation step is performed before dilution into ELISA buffer.

Suitably the denaturation step is performed after any protease treatment step.

AMPLIFICATION Some embodiments of the invention advantageously omit a prion protein amplification step. Indeed, it is a key feature of the invention that enrichment is used in order to increase the sensitivity of detection of PrPsc.

Nevertheless, amplification steps may be advantageously used in the methods of the present invention. Such amplification steps typically increase the sensitivity of the assays. In some embodiments of the invention, an enrichment step and amplification step are advantageously combined. By enriching before amplification, a greater input into the amplification procedure is advantageously obtained. This has numerous advantages such as cutting the time for amplification, and may lead to a reduced number of amplification rounds or steps.

A suitable amplification method is the protein misfolding cyclic amplification method (PMCA). This technique is well known in the art, and involves seeding the material to be amplified into a substrate comprising normal conformation PrP. This substrate may be constituted from recombinant proteins, or is more commonly constituted from brain homogenate of unaffected subjects. The misfolded material which is seeded into the amplification reagent then reacts with the normal PrP and catalyses its conversion into a misfolded form. The material is then typically sonicated to break up the aggregates of misfolded PrPsc, and a further addition of fresh PrP substrate material may then be made. By repeating the cycles, the amount of PrPsc misfolded material is amplified, enabling a more sensitive detection.

Other prion protein amplification methods may be used. For example, there are sonication free methods known in the art. These typically involve incubation in triton detergent based media and can lead to at least a five to ten fold enrichment. Although typically less powerful than PMCA based approaches, such methods may find application in the methods of the invention.

Thus invention relates to methods of detection of PrPsc, particularly to selective detection of PrPsc and/or selective purification or enrichment of PrPsc.

Suitably when the sample is blood, an amplification step is used.

FURTHER APPLICATIONS/ADVANTAGES

The invention also relates to a method of extracting or enriching PrPsc from a sample, said method comprising: (i) contacting said sample with a reagent which selectively binds PrPsc (ii) incubating said reagent with said sample (iii) separating or concentrating said reagent from said sample wherein said reagent comprises an ICSMlO antibody or a fragment or fusion thereof.

In another aspect, the invention relates to use of ICSMlO antibody or a fragment or flision thereof in the selective immunoprecipitation of PrPsc.

In another aspect, the invention relates to use of ICSMlO antibody or a fragment or fusion thereof in the ELISA selective detection of PrPsc.

In another aspect, the invention relates to use of ICSMlO antibody or a fragment or fusion thereof in the specific capture of PrPsc.

In another aspect, the invention relates to use of ICSMlO antibody or a fragment or fusion thereof in the enrichment of PrPsc in a sample.

The enrichment and/or capture steps have the further advantage of reducing reaction volumes relative to the same amount of material if it was assayed without enrichment/capture.

The enrichment/capture steps have the further advantage of reducing or eliminating inhibitors of amplification which can be present in blood.

The invention is now described by way of example, which is intended to be illustrative in nature rather than limiting the invention as defined in the appended claims. Reference is made to the following drawings.

In some embodiments capture/enrichment may be via sodium tungstate precipitation ('PTA' precipitation). Sodium tungstate precipitation is itself known. In these embodiments of the invention, i.e. when PTA is the capture/enrichment, then the invention further comprises an amplification step and suitably also further comprises ELISA detection.

Detection may be via FAST fluorescence, or by any other similar means. relying on fibril formation as a function of the amount of input material.

Brief Description of the Figures

Figure 1. Western blot detection of PrP from a range of species using monoclonal antibody ICSM33 Lane 1, 20 μl of 5% w/v uninfected mouse (CD-I) brain homogenate without PK digestion. Lane 2₅ 20 μl of PK-digested 5% w/v RML-infected mouse (CD-I) brain homogenate. Lane 3, 20 μl of 5% w/v uninfected, ovine brain homogenate without

PK digestion. Lane 4 20 μl of PK digested 5% w/v scrapie-infected sheep brain homogenate. Lane 5, 20 μl of 5% w/v uninfected, bovine brain homogenate without PK digestion. Lane 6, 20 μl of PK digested 5% w/v BSE-infected bovine brain homogenate. Lanes 7, 20 μl of 5% w/v uninfected, hamster brain homogenate without PK digestion. Lane 8 20 μl of PK digested 5% w/v Sc237- infected hamster brain homogenate. Lanes 9, 20 μl of 5% w/v normal human brain homogenate without PK digestion. Lane 10 20 μl of PK digested 5% w/v vCJD- infected brain homogenate. Uninfected control and prion infected brain homogenates are designated as U or I. The western blot was probed with biotinylated ICSM33 as primary antibody and developed by exposure to Kodak Biomax MR film. The positions of molecular mass markers are indicated in kilodaltons (kDa).

Figure 2. Immunoprecipitation of bovine, ovine and human PrP with ICSM33 is specific for PrP^Sc

(A) Lanes 1 and 2, western blot detection of PrP immunoprecipitated from uninfected normal bovine brain homogenate (U) by ICSM33 or ICSM35. Lanes 3 and 4, western blot detection of PrP immunoprecipitated from PK-digested BSE prion-infected brain homogenate (BSE) by ICSM33 or ICSM35. Lanes 5 and 6 contain the material immunoprecipitated from uninfected bovine brain or BSE prion-infected brain homogenate by the isotype control antibody BRIC126. (B) Lanes 1 and 2, western blot detection of PrP immunoprecipitated from control uninfected ovine brain homogenate (I) by ICSM33 or ICSM35. Lanes 3 and 4, western blot detection of PrP immunoprecipitated from PK-digested scrapie- infected ovine brain homogenate (I) by ICSM33 or ICSM35. Lanes 5 and 6, western blot detection of PrP immunoprecipitated from non-PK-treated scrapie- infected ovine brain homogenate by ICSM33 or ICSM35. Lanes 7 and 8 contain the material immunoprecipitated from uninfected ovine brain or scrapie-infected ovine brain homogenate by the isotype control antibody BRIC 126. (U) Lanes 1-3, western blot detection of PrP immunoprecipitated from control normal human brain homogenate by ICSM33 and ICSM35 and the isotype control antibody BRIC 126. Lanes 4-6, western blot detection of PrP immunoprecipitated from PK- digested sporadic CJD brain homogenate by ICSM33, ICSM35 and BRIC 126. Lanes 7-9, western blot detection of PrP immunoprecipitated from PK-digested vCJD brain homogenate by ICSM33, ICSM35 and BRIC126. The western blots were probed with biotinylated ICSM35 as primary antibody and developed by exposure to Kodak Biomax MR film. The positions of molecular mass markers are indicated in kilodaltons (kDa).

Figure 3. Determination of linear PrP epitopes that react with ICSM33

(A) ICSM33 was tested by peptide ELISA against 13 peptides, each 20 amino acids long and overlapping by 10 residues, covering the human PrP sequence between residues 91-231. The peptides were used at 10 μg/ml to coat ELISA plates. The results are expressed as the mean optical density of triplicate readings at 490nm with standard deviations from the mean superimposed as error bars.

(B) Fine epitope mapping of ICSM33 (IgG₁) was carried out using 10 peptides, each 15 amino acids long, overlapping by 13 residues, covering the human PrP sequence between residues 91-123. The peptides were used at 10 μg/ml to coat ELISA plates. Results are expressed as mean optical density of triplicate readings at 490nm with SD standard deviations from the mean superimposed as error bars. (C) Alignment of PrP amino acid sequences for the species tested with ICSM33. Residue 97 is highlighted in bold type and is responsible for the variable affinity of ICSM33 for PrP from different species.

Figure 4 Diagnostic immunoprecipitation of a blinded set of 20 brain homogenates with ICSM33

A total of 20 samples of brain homogenate comprising 6 homogenates from vCJD- infected brains and 14 from normal brains were blinded with the codes A through to T. All samples were treated equally and were subjected to immunoprecipitation with the monoclonal antibody ICSM33. The precipitated material was analysed directly by western blotting without pre-digestion with PK. The western blot was probed with biotinylated ICSM35 as primary antibody and developed by exposure to Kodak Biomax MR film. The positions of molecular mass markers are indicated in kilodaltons (kDa). vCJD samples were coded; A, C, G, J, N and R.

Figure 5 ICSM33 immunoprecipitates PK-sensitive isoforms of abnormal PrP.

(A) Five homogenates from vCJD-infected brains were treated equally and subjected to immunoprecipitation with the monoclonal antibody ICSM33. The precipitated material was analysed by western blotting both with and without pre- digestion with PK and thermolysin. The western blot was probed with biotinylated ICSMl 8 as primary antibody and developed by exposure to Kodak Biomax MR film. The positions of molecular mass markers are indicated in kilodaltons (kDa).

(B) The levels of detectable PrP in were quantified by densitometry of panel A. The results are displayed as a proportion of total abnormal PrP.

(C) Brain homogenates from patients with, inherited prion disease and control brain were treated equally and subjected to immunoprecipitation with the monoclonal antibody ICSM33. The precipitated material was analysed directly by western blotting without pre-digestion with PK. Western blots were probed with biotinylated ICSMl 8 as primary antibody and developed by exposure to Kodak Biomax MR film. The positions of molecular mass markers are indicated in kilodaltons (kDa). (D) Brain homogenates from three patients with the D178N mutation (B, C & D) and control brain (A) were treated equally and subjected to immunoprecipitation with the monoclonal antibody ICSM33. Sample B had previously yielded signal by standard western blot assay following pre-treatment with proteinase K; Samples C and D were negative by standard western blot examination. A known western blot positive D178N sample was run as a positive control in E (PK-digested) & F (no digestion). The precipitated material was analysed directly by western blotting without pre-digestion with PK. Western blots were probed with biotinylated ICSMl 8 as primary antibody and developed by exposure to Kodak Biomax MR film. The positions of molecular mass markers are indicated in kilodaltons (kDa). Homogenates analysed were negative following standard proteinase K treatment and high sensitivity western blotting.

Figures 6 to 10 show annotated antibody sequences. Note: yellow (boxed) residues are CDRs; grey shaded (unboxed, bold) is the start of the constant region; regions which are neither leader sequence, constant region nor CDR are defined as framework sequence.

Figure 11 shows a diagram. Figures 12, 13, 14, 15, 16 and 17 show western blots.

Figure 18 shows a diagram. Figures 19, 20, 21 and 22 show western blots.

Figure 23 shows a bar chart, figure 24 shows a diagram, figures 25 and 26 show bar charts.

Examples - Methods

Generation of monoclonal antibodies

Mouse monoclonal antibodies (mAbs) were produced in FVB/N Pm-p^m mice

(Zurich I,⁴²) immunised with either human alpha PrP^91"231 (α-PrP) or soluble beta PrP^91"231 (β-PrP)³²'⁴³ as previously described⁴⁴'⁴⁵. Briefly, hybridomas were selected after screening culture supernatants for levels of IgG production, then by ELISA using human recombinant α-and β-PrP^91"231 as antigens. They were further characterised by their ability to bind native PrP in U937 and NSO cells by flow cytometry (FACS analysis) and to normal and PK-treated diseased brain homogenates by dot-blot and by immuno-precipitation. Binding to denatured PrP was assessed by western blotting to PrP^c and PK-treated PrP^So derived from normal control and diseased brain homogenates respectively. The epitopes of the mAbs were mapped by peptide ELISA and their isotypes were determined, using proprietary reagents (Perbio Science or Roche Biochemicals). ICSM 33 and ICSM35 are both IgG_2b mouse imrnunoglobins.

Tissue samples and homogenate preparation

For characterisation of mAbs, homogenates from human, hamster, mouse, ovine and bovine brain were used. Human brain frontal cortex homogenates were prepared from normal and vCJD-affected individuals as 10% w/v preparation in Dulbecco's phosphate-buffered saline (PBS) lacking calcium and magnesium ions using a Dounce homogeniser. Whole mouse brain from normal CDl mice or terminal RML-infected CDl mice were prepared as 20% w/v homogenates using a Ribolyser (Hybaid). Whole hamster brain from normal Syrian hamsters of terminal Sc237-infected Syrian hamsters were prepared similarly. Medulla oblongata from normal or natural-scrapie infected sheep and cerebral cortex derived from normal or BSE infected cattle were also prepared using a Ribolyser. Homogenates were stored as aliquots at -80°C.

Protease Treatment

Proteinase K (EC 3.4.21.14) from Tritirachium album limber was obtained freeze- dried from Merck Biosciences, Ltd, Nottingham, UK. The specific enzymatic activity is approximately 30 Anson units / g, where 1 Anson unit is the amount of enzyme that liberates 1 mmol of FoKn positive amino acids / min at pH 7.5 and 35

⁰C using haemoglobin as substrate. Diseased brain homogenates were treated with

Proteinase K (PK) for 60 min at 37°C at concentrations of; 40 μg/ml PK for mouse brain, 50 μg/ml for human brain and at 100 μg/ml of PK for sheep and cattle brain homogenates. Thermolysin (EC 3.4.24.27) from Bacillus thermoproteolyticus rokko was obtained freeze-dried from Sigma-Aldrich, St Louis, MO, USA. The specific enzymatic activity is 50-100 units / mg protein where 1 unit liberates 1 μmole of tyrosine / min at pH 7.5 and 37 ⁰C using casein as substrate. Digestions, with thermolysin were carried out at lOOμg/ml for 1 hour at 70°C. Proteolysis reactions were stopped by the addition of 4-(2-aminoethyl)-benzene sulfonyl fluoride (AEBSF) (Roche, Lewes, UK) to a final concentration of 1OmM.

Western Blot Analyses

Samples were analysed as previously described ¹⁶. The membranes were incubated with anti-PrP monoclonal antibody, either ICSM33 or biotinylated ICSM35 (D- Gen Ltd, London, UK) diluted to 0.2 μg/ml in PBST for at least 60 min before washing in PBST (30 min). Blots probed with ICSM33 were incubated for one hour at room temperature with a 1:10,000 dilution of goat anti -mouse IgG-alkaline phosphatase conjugate (Sigma) in PBST. Blots probed with biotinylated ICSM35 were similarly incubated with a streptavidin-alkaline-phosphatase conjugate (DAKO UK Ltd) diluted into PBST according to the manufacturers instructions. Membranes were washed for a total of 60 min with PBST and twice for 5 min with 2OmM Tris pH 9.8 containing ImM MgCl₂ (1 x assay buffer; Tropix me) before development in chemiluminescent substrate (CDP-Star; Tropix Inc) and visualisation on Biomax MR Film (Kodak).

Immunoprecipitation of brain homogenates

Brain homogenates were diluted to 5% w/v in 2% sodium lauroylsarcόsine, followed by adjustment with 250U/ml benzonase and incubated at 37°C for 30 min. Control normal brain homogenates were centrifuged at 100 x g for 2 min and the supernatant retained and treated with a protease inhibitor cocktail (Protease Inhibitor Complete, Roche Biochemicals). Diseased brain homogenates were PK digested and treated with AEBSF as described above. Prior to immune- precipitation, all homogenates were diluted to 0.5% w/v homogenate with PBS. These samples were then adjusted with an equal volume of culture medium (RPMI, 10% v/v FCS) containing 10 μg/ml of the appropriate anti PrP IgG and incubated for 1-2 hours on a rotator at room temperature. 25 μl of Protein-G Dynabeads (with a binding capacity of ~8-12 μg of IgG) (Dynal Biotech, UK) was then added to the samples followed by incubation for 18 hours at 4°C on a rotator. Magnetic beads were subsequently recovered using a magnetic tube rack and the beads were washed five times for 5 min with PBS 2% v/v Tween-20, 2% w/v NP40 on the rotator. Washed beads were resuspended in an appropriate aliquot of 2 x SDS loading buffer and the samples heated to 100°C for 10 min, Beads were sedimented by centrifugation at 15,000 x g for 2 min prior to analysis of the supernatant by western blotting, hi some experiments, the final wash steps were omitted and magnetic beads containing immune-complexes were processed directly for western blotting.

Peptide and recombinant PrP ELISA

For epitope mapping, peptides comprising 20 amino acids were synthesised that span the human PrP sequence from position 91 to 231 with a 10 residue overlap between each sequential peptide, In some experiments 15mer peptides with 13 amino acid residue overlap were also used.

Synthetic peptides or recombinant murine PrP^91"231 (50 μl of a 10 μg/ml peptide solution dissolved in 35mM NaHCO₃, 15mM Na2CO₃ pH 9.6) were absorbed to both high or medium binding, 96 well plates (Greiner) for 1 hour at 37°C. The plates were then washed four times with PBS + 0.05% v/v Tween-20 (PBST) using a plate washer (Well wash 4- Thermo electron corporation, UK). Non-specific binding was blocked with RPMI (Gibco, Paisley, UK) containing 10% v/v foetal calf serum (RFlO) for 1 hour at room temperature. Plates were washed once with PBST prior to incubation with 50 μl 10 μg/ml ICSM33 in PBST for 2 hours at 37°C. The plates were then washed 3 times with PBST prior to the addition a 1/1000 dilution of horseradish-peroxidase (HRP) conjugated anti-mouse IgG (Sigma, UK) in PBST for 1 hour at 37°C. The plates were again washed 4 times with PBST and developed by addition of substrate (o-phenyldiamine) (Sigma, UK). Reactions were stopped by addition of 50 μl 3 M sulphuric acid prior to spectrophotometric analysis at 490 nm.

Specific inimunoprecipitation of PrP*⁰ from CJD and FFI brain without protease treatment 20 μl of 10% w/v brain homogenate in PBS was adjusted with an equal volume of 2 x capture buffer (10OmM Tris.Cl pH 8.4 containing 5% w/v sodium lauroylsarcosine, 5% v/v Triton X-100, 5% w/v bovine serum albumin (BSA) and 10 x Protease Inhibitor Complete cocktail (Roche). Samples were then diluted with I x capture buffer containing ICSM33 to give a final volume of 200 μl and a final IgG concentration of 3μg/ml. The samples were the incubated on a rotator for 18 hours at 4°C after which 25 μl of Protein- A Dynabeads (with a binding capacity of ~6μg of IgG) were added and the samples incubated at room temperature for 3 hours in a rotator. Magnetic beads were washed four times for 5 min with 250 μl of capture buffer using a magnetic tube rack and then similarly washed four times with 250 μl PBS containing 2% v/v Tween-20 and.2% w/v NP40, followed by a final wash with 250 μl PBS. Washed beads were resuspended in 25 μl of PBS and 25 μl of 2 x SDS-loading buffer and heated to 100°C for 10 min. Beads were sedimented by centrifugation at 15,000 x g for 2 min prior to analysis of the supernatant by western, blotting.

Example 1 : Molecular diagnosis of human prion disease using an antibody specific for PrP^Sc

1. introduction

Variant Creutzfeldt-Jakob disease (vCJD) is a devastating and uniformly fatal prion disease. Its causal association with bovine spongiform encephalopathy has raised concerns of a significant human epidemic. The existing World Health Organisation

(WHO) diagnostic criteria, whilst useful for epidemiological purposes, do not facilitate early disease diagnosis and a definitive diagnosis can be achieved only be means of neuropathological examination. The prolonged asymptomatic incubation period and possibility of carrier states together with recent recognition of transfusion-associated iatrogenic vCJD pose major public health concerns and have added impetus for development of a blood based molecular diagnostic

. .1.2,3 test .

Figure 11 shows a diagram of the need for a simple and sensitive molecular diagnostic test for vCJD .

Challenges associated with development of a molecular diagnostic test

The key marker of vCJD infection is deposition of PrP^Sc a conformational^ altered isoform of the normal prion protein, PrP^c. Development of screening tests has proved challenging due to the large excess of PrP^c over the disease-associated

PrP^Sc, both of which are usually detected in immunoassays. Established diagnostic tests depend upon protease pre-treatment of tissue samples to degrade background PrP^c, with a concomitant loss of over 50% of disease associated PrP^Sc. Despite concerted efforts, no effective diagnostic test has been developed based upon on antibody with selectivity for PrP^Sc. Using a novel conformational state of recombinant human PrP (β-PrP) we have developed a monoclonal antibody, ICSM 33, with selectivity for disease-associated isoforms of PrP⁵. PrP^c is loosely bound and removed after minimal washing, whereas aggregated PrP^Sc remains tightly bound via multivalent interactions. Exploitation of this characteristic for detection of PrP^Sc was investigated.

2. Methods

Brain tissue was obtained at autopsy from control and histopathologically confirmed vCJD individuals with consent for research use. Twenty brain homogenates were analysed by immunoprecipitation with ICSM 33 blind to disease status. Immunoreactivity was determined after electrophoresis and high sensitivity western blot analysis. In addition, whole blood samples spiked with control and vCJD brain tissue were assayed using optimised ICSM 33 conjugated beads. In order to determine the sensitivity of detection of PrP^Sc using this technique, increasing volumes of control blood was assayed after spiking with a vCJD brain dilution series (prepared by spiking vCJD brain homogenate into control brain homogenate). The optimised protocol has been applied to assay of clinical samples from vCJD patients including whole blood, tonsil and rectal tissue.

Probing was performed with anti-PrP monoclonal antibody ICSM 18B in conjunction with alkaline phosphatase-conjugated secondary reagents.

^' Immunoreactivity was reported by an independent observer following development with chemiluminescenf substrate and visualisation on Kodak Biomax film.

3. Results

Figure 12 shows selective discrimination between control and vCJD brain homogenate by detectionof PrP^Sc following overnight immunoprecipitation with ICSM 33 and high sensitivity western blot PrP^Sc

In a blinded series, all vCJD brain homogenates produced clearly visible immunoreactivity and no signal was produced by any control homogenates.

Figure 13 shows high sensitivity western blot detection of PrP^Sc from vCJD brain homogenate following overnight immunoprecipitation with ICSM 33 and detection with anti-PrP mAb ICSM 18B. Clear immunoreactivity is demonstrated for all vCJD brain homogenates (A, C, G, J, N,R).No immunoreactivity is detected for control brain homogenates (B, D, E, F, H, I, K, L M, O, P, Q, S, T)

Immunoprecipitation with ICSM 33 successfully discriminates between whole blood samples spiked with control and vCJD brain (figure 14). Figure 14 shows selective detection of PrP^Sc from whole blood spiked with vCJD brainhomogenate following overnight immunoprecipiation with ICSM 33and high sensitivity western blot

Detection is possible up to a dilution of lOOnl 10% w/v vCJD brain homogenate spiked into 8mls of whole blood. Figure 15 shows immunoprecipitation of 8mls whole blood spiked with vCJD brain dilution series using 1CSM33. Figure 16 shows 4mls of blood from α pαfient wifh neuropαfhologicαlly proven vCJD was subjected to immunoprecipitation with ICSM33. lmmunoprecipitated material was analysed by high sensitivity western blotting with no protease pre- treatment

The method successfully detects disease-associated PrP in peripheral tissues from vCJD patients including tonsil and rectum (figure 17). Figure 17 shows high sensitivity western blot following immunoprecipitation with ICSM 33 of: A: vCJD rectal tissue and B: vCJD tonsil tissue homogenate

4. Conclusions

A robust technique of immunoprecipitation using the novel monoclonal antibody ICSM 33 has been optimised allowing selective detection of disease associated PrP. This technique may be employed for the detection of small quantities of PrP^Sc in tissue samples and obviates the requirement for protease pre-treatment. The technique enables concentration and detection of up to 10OnI 10% w/v vCJD brain homogenate spiked into 8mls of whole blood (figures 14 & 15) and disease- associated PrP in peripheral tissues from vCJD patients including tonsil and rectum (figure 17). The sensitivity of this method whilst coupled with high sensitivity western blot remains below the threshold required for detection of PrP^Sc in vCJD blood samples (figure 16).. The challenge remains to further develop the technique for use in conjunction with other techniques currently being investigated for increasing the limit of PrP^Sc detection in blood such as PMCA (see other examples) and novel sandwich ELISA (see other examples).

References

Llewelyn CA. ei al.. Lancet 363 (2004): 417-21 Peden A.H. ef a/. Lancet 364 (2004): 527-29 Wroe SJ. eϊal. Lancet 368 (2006): 2061-2067 Jackson G.S. ef a/. Science 283 (1999): 1935-37 Khalili-Shirazi A., et al. The Journal of Immunology 174 (2005): 3256- 63 Example 2: Discrimination between prion-infected and normal blood samples by PMCA at a pre-clinical stage.

1. Background . The development of sensitive methods for blood-based diagnostics and screening is an urgent, national strategic priority. The potential for a 'self-sustaining epidemic of secondary vCJD infection from contaminated blood remains a possibility despite the introduction of leukodepletion. In addition to the known risk of vCJD transmission via blood transfusion the potential for infection from contaminated processed blood products exists with over 6,000 patients identified who have received processed products known to be contaminated with vCJD prions. Figure 18 shows a diagram helping to illustrate this.

Diagnosis of prion infection from blood samples requires the detection of extremely low quantities of PrPSc. Protein Misfolding Cyclic Amplification (PMCA) is a technique which can amplify small amounts of seed PrPSc to a level detectable by conventional methodsl . PMCA of buffy coat fractions derived from scrapie infected Hamster blood have indicated that PrPSc is detectable in pre- and post- symptomatic animals. Diagnostic discrimination between prion-infected and normal samples has yet to be shown in whole blood. Application of PMCA to the molecular diagnostic testing of blood samples may enhance the ability to detect PrPSc in blood and allow ante-mortem detection of prion infection.

Figure 19 shows RML infection time course in CD-I mice. CD-I mice were inoculated i.e. with 30μl of a 1% (w/v) RML-infected brain homόgenate. Groups of

20 mice were culled at 0, 20, 40, 60, 80, 100, 120, 140 after infection and succumbed to clinical disease at 148 days after infection. Brain, spleen and blood were collected at each time point. Brains were analysed for the presence of PrPSc by western blotting and infectivity quantified by Scrapie Cell Assay (ref.5). Western blotting of brain homogenates gave uniformly positive results from 120 days post infection and levels of infectivity reached a maximum at around 140 days.

2. Aims

To use the PMCA methodology to amplify PK resistant PrP (PrP^RES) from whole blood collected from RML infected mice and to robustly differentiate between prion- infected and uninfected blood samples.

3. Methods

PMCA was performed as described previously⁴. Brain homogenate or whole ^■ blood was diluted into PMCA substrate brain homogenate and subjected to multiple cycles of PMCA. Serial PMCA (sPMCA) involved repeated rounds of amplification following re-dilution into fresh substrate. All samples were analysed for the presence of PrP^RES by western blotting.

4. Results

Figure 19 shows serial PMCA of RML brain homogenate. As a proof of concept, 10% (w/v) RML-infected brain homogenate was diluted 1 :400 into PMCA substrate containing brain homogenate from uninfected CD-I mice and subjected to 140 rounds of amplification (sPMCA #1). Samples were then diluted into fresh substrate and PMCA was repeated (sPMCA #2. and #3). Amplification of PrP^RES is in the region of 20-40 fold per cycle of sPMCA giving a total amplification of ~16,000x from the original RML dilution.

Figure 20 shows RML infection time course in CD-I mice.CD-1 mice were inoculated i.e. with 30μl of a 1 % (w/v) RML-infected brain homogenate. Groups of 20 mice were culled at 0, 20, 40, 60, 80, 100, 120, 140 after infection and succumbed to clinical disease at 148 days after infection. Brain, spleen and blood were collected at each time point. Brains were analysed for th presence of PrPSc by western blotting and infectivity quantified by Scrapie Cell Assay5. Western blotting of brain homogenates gave uniformly positive results from 120 days post infection and levels of infectivity reached a^' maximum at around 140 days.

Figure 21 shows Serial PMCA can differentiate RML-infected from normal whole blood.

lμl of whole blood from infected RML CD-I mice collected 140 days post infection was diluted 1:100 into PMCA substrate homogenate. Normal, uninfected whole blood and whole blood spiked with RML (1:10) were also diluted 1:100 into PMCA substrate as negative and positive controls respectively. Serial PMCA was performed on all samples. After 4 consecutive rounds of sPMCA, amplification of PrPRES could be detected in all of the RML infected blood samples. No PrPRES is detectable in the normal uninfected blood samples.

5 Figure 22 shows α diagram of strategy in molecular diagnostic approaches to Prion disease.

5. Conclusions. Serial PMCA performed on small volumes of whole blood samples (lμl) can amplify PrPRES to readily detectable levels. The very high sensitivity

10 achieved allows animals infected with RML to be accurately diagnosed at a pre- symptomatic stage of disease for the first time. Future work will focus upon coupling sPMCA with a pre-enrichment step for disease-associated PrP by imrnunoprecipitation with a PrPSc-specific antibody (poster P03.144and detection with a high sensitivity ELISA assay (poster PO 1.59). Figure 4 predicts a scenario in

15 which disease-associated PrP is concentrated from a large volume of whole blood (up to 1 OmIs), amplified by sPMCA and then detected with a high sensitivity ELISA that does not require PK.

References:

20 1. Soto C. et al. FEBS Lett. 2005: 579, 638-642.

2. Cαstillα J. et al. Nat. Med. 2005: 11, 982-985.

3. Sαά P. ef ah. Science 2006: 313, 92-44.

4. Cαstillα J. et al. Methods Enzymol. 2006: 412, 3-21.

5. Klδhn P.C. ef al. PNAS 2003: 100, 1 1666-7} . •25

Example 3: A highly sensitive and specific ELISA for the determination of prion infection in human samples without the use of proteinase K.

1. Introduction

5 A highly sensitive assay for the detection of PrP^Sc is vital for the early diagnosis of prion disease, as well for the screening of blood and organ donations. Currently the best method for diagnosing prion disease is the detection by western blotting of PrP^Sc in biopsied tissue which requires digestion with proteinase K (PK) to distinguish between PrP^c and PrP^Sc. However, besides removing PrP^c, it now 10 appears that the majority of disease related PrP may be destroyed by PK under conditions that are typically employed to detect prototypical PrP^Sc1'². New methods that do not require PK can extend the sensitivity of abnormal PrP detection and will assist in the development of rapid, blood-based diagnostic tests. Using a combination of limited thermolysin digestion and PrP^Sc selective 15 antibodies^3"5 we have developed a highly sensitive assay for the detection of PrP^Sc in whole blood.

2. Objecf/ves

The main objective was to increase the sensitivity and throughput of immunoassays for the detection of PrP^Sc in blood. In particular, to identify

20 conditions for the effective discrimination of PrP^Sc from PrP^c without the need for PK digestion. Conditions were optimised for the detection of vCJD brain homogenate spiked into whole blood from unaffected donors to allow for the use of any blood- based substrate. An additional important objective was to ensure the resulting ELISA could be coupled to other diagnostic strategies to further increase sensitivity.

25 VVe have successfully integrated the ELISA with methods for the immunoprecipitation of PrP^Sc from large volumes of whole blood.

3. Methods vCJD brain homogenate, diluted into whole blood obtained from anonymous, unaffected donors, was analysed by enzyme-linked immunosorbent assay (ELISA). 30. Specifically, samples were treated with thermolysin before being denatured and diluted into ELISA buffer for antigen presentation. PrP^Sc was selectively captured by ICSMlO and detected with biotinylated ICSM35. In experiments where immunoprecipitation and ELISA were combined, PrP^Sc was recovered by from vCJD brain homogenate spiked into 250μl or 8ml of whole blood (for further details see previous examples) before detection by ELISA. Antibodies and buffers are supplied by D-Gen Ltd*

4. Results Figure 23 shows sensitivity of ELISA to vCJD serially diluted into normal whole blood. vCJD brain homogenate (10% w/v) was serially diluted into lOμl uninfected whole blood. Samples were prepared for assay and diluted to 200μl with ELISA buffer and 50μl added to each well (n=3). Normal uninfected control sample was treated identically and is an average of 4 normal samples (n=3 per sample). Cut-off for detection is the averaged normal background + 3 Standard Deviations [SD) .Leve/s as low as InI can be distinguished from normal samples.

Figure 24 shows combined I.P./ELISA assay. PrP^Sc in whole blood is selectively captured by ICSM33 coated magnetic beads. The beads are washed to remove PrP^c. Thermolysin treatment is performed while PrP^Sc is still bound to the beads. Antigen is eluted in denaturing conditions and applied to ICSMlO coated ELISA plates for detection.

Figure 25 shows sensitivity of assay to vCJD serially diluted into uninfected whole blood. vCJD brain homogenate (10% w/v) was serially diluted into 250μl uninfected whole blood. PrP^Sc was selectively recovered by I. P. with ICSM33, thermolysin treated and eluted in denaturing conditions. ELlSA buffer was added (1 :20 dilution) and 50μl analysed per well (n=3). Normal control blood was treated identically and is an average of 3 samples (n=3 per sample). The cut-off for detection was set at the mean normal background + 3 SDs. The combined I.P./ELISA methodology allowed detection of PrP^Sc to 4nl 10% (w/v) vCJD homogenate per assay well from a starting volume of 250μl of whole blood, which is equivalent to 2 pg of PrP^Sc. Figure 26 shows sensitivity of assay to vCJD diluted into 8ml uninfected whole blood.10% (w/v) vCJD brain homogenate was spiked into 8ml of normal whole blood.Following specific capture of PrP^Sc with ICSM33 coated beads, thermolysin • treatment was performed and the antigen eluted in a minimal volume. ELISA buffer was added (1 :20 dilution) and^'50μl loaded per ELISA well (n=3). 8ml Normal control blood was treated identically (50μl per well, n=3). Again the cut-off for detection was sei at the mean ^'+ 3 SDs of the normal background. PrP^Sc was detectable at 3OnI 10% (w/v) vCJD brain homogenate per assay well (-15 pg). This is equivalent to detection of vCJD brain at >2.5 million fold dilution.

5. Conclusions

• We have developed an assay which allows the rapid, sensitive and selective detection of PrP^Sc at biologically relevant levels. • The assay does not require PK digestion for discrimination between PrP^c and PrP^Sc.

• Detection of PrP^Sc from vCJD brain homogenate diluted into whole blood is in the picogram range.

• Coupled with selective capture of PrP^Sc from amenable volumes of blood, allows detection at a level equivalent to a 2.5 million fold dilution of brain.

• Thus the combined I.P./ELISA approach provides a robust, sensitive and rapid method for the detection of extremely small quantities of PrP^Sc from clinically relevant volumes of whole blood.

References: .

1. Safar, J.G. et a/. PNAS 2005: 102, 3501-3506.

2. Thackray A.M. et a/. Biochem. J 2007: 401, 475-483.

3. Owen, J. P. et a/. Mo/ Biotechnol. 2007: 35:161-170

4. Khalili-Shirazi, A. et a/. J Gen Virol. 2005: 86, 2635-2644. 5. Khalili-Shirazi, A. et ol. BBA 2007: manuscript accepted, online Sept. 2007.

6. D-Gen Limited is an academic spin-out company working in the field of prion disease diagnosis, decontamination, and therapeutics. D-Gen markets the ICSMlO, ICSM35 and ICSM33 antibodies used in this study. Example 4: Molecular diagnosis of vCJD using a monoclonal antibody specific for disease-associated PrP

This example illustrates uses of an antibody specific for PrP^Sc

Summary

We describe an antibody, raised against a β-PrP isoform, that is specific for disease-associated PrP and has been used in the diagnosis of vCJD without the need for PrP^c depletion by proteolysis or other methods. The methods described are capable of detecting twice the amount of abnormal PrP that can be seen using conventional PK-based approaches. This facilitates the sensitive detection of PrP^Sc in peripheral tissues or fluids, particularly where the ratio of PrP^c to PrP^Sc is high and/or proteolysis does not provide sufficient discrimination.

Using the novel conformational recombinant human PrP, β-PrP, which has several physico-chemical properties in common with PrP^{Sc 32} we have isolated a unique monoclonal IgG molecule, ICSM33, with strong selectivity for PrP^Sc. The affinity for PrP^Sc is such that that antibody can be used within suitable protocols here described as a practical diagnostic assay for vCJD that does not require proteolysis. This immunoassay can be used to detect disease-associated PrP in patient samples where conventional western blotting cannot. Without wishing to be bound by theory, this effect is presumably due to the relative protease sensitivity of the aberrant PrP.

Western blot detection of PrP with ICSM33 reveals species-specific differences.

The use of ICSM33 as primary antibody for the western blot detection of denatured PrP from a variety of mammals revealed a striking specificity for certain species (Figure 1). Whilst PrP from both Syrian hamster and CD-I mice (expressing Prnp allele a) could be readily detected there was no observable PrP signal from ovine, bovine or human brain homogenates. This pattern of immunoreactivity was observed with both normal and prion-infected brain homogenates from these species. Following proteolytic digestion with PK the expected ratio of PrP^Sc glycoforms associated with RML and Sc237 prion strains was observed (Figure 1).

ICSM33 selectively immunoprecipitates PrP⁵⁰ from several species. To further investigate the reactivity of ICSM33 towards native PrP isoforms, brain homogenates from a range of species were subjected to immunoprecipitation followed by western blot detection with ICSM35. Both PrP^c and PrP^Sc were effectively immunoprecipitated from murine and hamster brain homogenates. However, with PrP from species that did not react with ICSM33 by direct western blotting (Figure 1) a disease-specific pattern of immunoprecipitation was observed in which PrP^So, but not PrP^c, was efficiently recovered (Figure 2). Immunoprecipitation of homogenates prepared from human sporadic CJD brain with type 1 PrP^Se or vCJD brain with type 4 PrP^Sc (according to classification of Hill et al³³) yielded glycoforms typical of the respective CJD sub-types ^2>33'³⁴. Strikingly, ICSM33 failed to precipitate PrP^c from uninfected, normal human brain homogenates. Both positive and negative control antibodies behaved as expected; with ICSM35 immunoprecipitating PrP from normal and infected brain, whereas the isotype control, BRIC 126 was consistently negative. A similar disease-specific pattern of immunoprecipitation was observed with bovine and ovine brain homogenates. Brain homogenates infected with BSE or scrapie yielded distinct PrP glycotypes whereas irnmunoprecipitations from normal bovine and ovine homogenates were negative.

ICSM33 recognises a linear PrP epitope between the octapeptide repeat region and the C-terminal globular domain

The observation of disease-specificity by immunoprecipitation suggests ICSM33 may be able to differentiate conformational differences between native PrP^c and PrP^Sc. However, such a hypothesis is difficult to reconcile with the species specificity observed by western blotting which probes denatured PrP. In order to clarify the nature of PrP recognition by ICSM33 we defined the linear binding epitope by standard peptide ELISA against a panel of overlapping peptides (Figure 3A&B) that encompass the human PrP sequence from residues 91-231 (against which the antibody was originally raised). The antibody was found to bind strongly to a linear sequence of 15 amino acids containing residues 93-107. Within this linear epitope there is only one polymorphic residue at position 97 (Figure 3C) that varies in accordance with the species specificity observed in Figure 1 and is required to be asparagine for high affinity binding and thus recognition of denatured PrP by western blotting.

ICSM33 selectivity for human, bovine and ovine PrP^^c results from multivalent binding to PrP^⁰ aggregates. Under the detergent conditions used for immunoprecipitation (treatment with sodium lauroylsarcosine) human PrP⁰ is soluble (and presumably monomelic) whereas PrP^Sc exists as insoluble aggregates¹⁶'³⁵. PrP^Sc polymers may therefore be bound multivalently by IgG-protein G beads whereas PrP^c monomers (that are not physically linked) can only be bound monovalently by a single antigen binding site on the IgG molecule. This difference in valency will dramatically affect the apparent dissociation constants for IgG binding to PrP⁰ or PrP^Sc. To assess the contribution of multivalent interactions between ICSM33 and PrP isoforms we performed immunoprecipitation reactions in which the captured immune complexes were analysed essentially at equilibrium without washing. Under these conditions we found that ICSM33 was capable of binding to and precipitating PrP⁰ from human, ovine and bovine homogenates. However, unlike PrP^Sc polymers from these species, that are retained after washing the immune complexes, PrP⁰ was readily removed by brief washing indicating a much lower binding affinity: These collective data, combined with the polymorphism of PrP at residue 97 provides a potential molecular basis for the observed species and PrP isoform selectivity of ICSM33. For mouse and hamster PrP, the presence of asparagine at residue 97 appears to confer a binding affinity high enough for ICSM33 to recognize denatured PrP on western blots and efficiently capture and retain both PrP⁰ and PrP^Sc from brain homogenate. In the case of human, bovine and ovine PrP variance at residue 97 causes a reduction in binding affinity of ICSM33 resulting in an inability to detect denatured PrP on western blots and the ability to only capture and retain PrP^Sc with high affinity due to multivalent interactions. vCJD can be diagnosed using ICSM33 without the use of protease (proteinase K)

To assess the utility of ICSM33 in the diagnosis of vCJD we prepared a panel of twenty brain homogenates of which fourteen were normal human brain and six were prepared from vCJD infected brain. The homogenates were coded and subject to immunoprecipitation in a blinded manner. Following the detection of precipitated PrP by western blotting (Figure 4) all six vCJD cases were unequivocally identified by the presence of PrP bands in lanes A, C, G, J, N and R.

No background bands were detected in the immunoprecipitations performed from normal brain homogenates.

Immunoprecipitation of disease-associated PrP from vCJD brain homogenate reveals the presence of variable but significant proportion of PK sensitive material Samples of brain homogenate from five individual cases of vCJD were subjected to immunoprecipitation with ICSM33. The recovered material was tested for resistance to proteolysis by digesting with either proteinase K or thermolysin and visualised by western blotting (Figure 5A). The proportion of PrP remaining was determined by densitometry and the results are displayed in Figure 5B. In agreement with recent reports thermolysin does not significantly degrade disease- associated PrP ³⁶'³⁷, producing a mean reduction in intensity for the five cases of only 11%. hi contrast PK digestion destroys almost half (44%) of the disease- associated PrP that can be recovered by ICSM33.

Diagnostic immunoprecipitation of brain homogenates from patients with inherited prion disease.

Brain homogenates from patients with inherited prion disease and control brain were treated equally and subjected to immunoprecipitation with the monoclonal antibody ICSM33 before visualisation by western blotting (Figure 5C). There was clear distinction between affected individuals with a range of inherited forms of prion disease and control, with the level of disease-associated PrP associated with a case carrying a D178N mutation being particularly high. Three further cases of inherited prion disease associated with D178N mutations were analysed by immunoprecipitation and western blotting in Figure 5D. All three cases had previously been analysed by western blotting and immunohistochemistry (IHC). Although IHC had confirmed the presence of abnormal PrP in all cases, two of these (C and D) had been negative for PrP by conventional western blotting involving the use of PK. Immunoprecipitation with ICSM33 resulted in the detection of abnormal PrP in all cases, clearly demonstrating the benefits of a PrP^Sc selective antibody over and above the use of PK-based methods.

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^' I'

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43. Jackson,G.S., Hill,A.F., Joseph,C, Hosszu,L.L.P., CIarke,A.R. & Collinge,J. Multiple folding pathways for heterologously expressed human prion protein. Biochimica et Biophysica Acta 1431, 1-13 (1999).

44. Khalili-ShirazijA., Quaratino,S., Londei,M. et al Protein conformation significantly influences immune responses to prion protein. J Immunol 174, 3256-3263 (2005).

45. Khalili-Shirazi,A., Summers,L., Linehan,J. et al PrP glycoforms are associated in a strain-specific ratio in native PrPSc. J Gen Virol 86, 2635- 2644 (2005). AU publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described antibodies, methods and uses of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.

Sequence Listing

SEQ ID NO: 1 - ICSM35VH

ATGGAATGGACCTGGGTCATTCTCTTCCTCCTGTCAGTAACTGAAGGTGT CCACTCCCAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGΆAGCCTG

GGGCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTACACATtCAGTAAC TCCTGGATGAACTGGGTGAAGCAGAGGCCTGGGAAAGGTCTTGAgTGGAT TGGACGGATTTATCCTGAATATGGACATGCTGACTACAATGGGAAGTTCG AAGGCAAGGCCACACTGACTGCTGACAGATCCTCCAGCACAGCCTACATG CACCTCAGCAGCCTGACGTCTGAGGACTCTGCGGTCTACTTCTGTGCACG AGCCCCACTACGGTACCCCTACTTTGACTACTGGGGCCAAGGCACCACTC TCACAGTCTCCTCA

SEQ ID NO: 2 - ICSM35VK

ATGGTGTCCACAGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGG TACCAGATGTGATATCCAGATGACaCAGAcTTCATCCTCCCTGTCTGCCT CTCTGGGAGACAGAGTCTCCATCAGTTGCAGGGCAAGTCAGGACATTTCC AATTATTTAAACTGGTATCAGCAGAaACCAGATGGAACTGTTAAACTCCT. GATCCACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTtCAGTG ^•GCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCCACCTGGAGGAA GAAGATATTGCCACTTACTTTTGCCAACAGGGTAATGCGcTTCCTCCGAC GTTCGGTGGCGGCACCAAGCTGGAAATCAAA ^'

SEQ ID NO: 3 - ICSM18VH

ATGGAATGGAGCTGGGTTTTCCTCTTCCTCCTGTCAGGAACTGCAGGTGT CCTCTCTGAGGTCCAGCTACAACAGTCTGGACCTGAGCTGGTGAAGCCTG

GGTCTTCAGTGAAgATATCCTGCAAGGCATCTAGAAACACATTCACTGAC

TATAACTTGGACTGGGTGAAGCAGAGCCATGGAAAGACACTTGAGTGGAT

TGGAAATGTTTATCCTAACAATGGTGTTACTGGCTACAACCAgAAgTTCA

GGGGTAAGGCCACACTGACTGTAgACAAGTCCTCCAGCACAGCCTACATG GAGCTCCACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCCCT

TTATTACTACgATgTCTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCT

CTGCA SEQ ID NO : 4 - ICSM181C

ATGGATTTACAGGTGCAGATTATCAGCTTCCTGCTAATCAGTGCCTCAGT CATAATATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGT CTGCATCTCCAGGGGAGAA_GGTCACCATGACCTGCAGTGCCAGCTCAAGT GTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAG ATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCA GTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTATGGAG GCTGAAGATGCTGCCACTTATTTCTGCCACCAGTGGAGAAGTAACCCATA CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCAC CAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGT GCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGT CAΆGTGGAAGATTGATGGCAGTGAACGACAAAΆTGGCGTCCTGAACAGTT GGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTC ACGTTGACCAAGGACGAGTATGAΆCGACATAACAGCTATACCTGTGAGGC CACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAG AGTGTTAGTGA

Claims

1. A method of extracting or enriching PrPsc from a sample, said method comprising:

(i) contacting said sample with a reagent which selectively binds PrPsc (ii) incubating said reagent with said sample

(iii) separating or concentrating said reagent from said sample wherein said reagent comprises an ICSM33 antibody or a fragment or fusion thereof.

2. A method of extracting or enriching PrPsc from a sample according to claim 1 further comprising dilution of said sample with buffer comprising detergent in an amount effective for cell membrane disruption and comprising protein in an amount effective to ameliorate non-specific binding of reagents.

3. A method according to claim 1 or claim 2 wherein separating said PrP bound reagent comprises capturing said antibody or fragment or fusion thereof by attachment to a solid phase substrate.

4. A method for amplification of PrPsc, said method comprising extracting or enriching PrPsc according to any of claims 1 to 3, and then amplifying said extracted or enriched PrPsc.

5. A method according to claim 4 wherein, amplification is by protein misfolding cyclic amplification (PMCA). ' .

6. A method for detection of PrPsc in a sample, said method comprising extracting or enriching PrPsc according to any of claims 1 to 5, optionally amplifying said PrPsc, and detecting said PrPsc by ELISA.

7. A method for detection of. PrPsc in a sample, said method comprising detecting said PrPsc by ELISA, wherein detection comprises dilution with buffer comprising detergent in an amount effective for cell membrane disruption and protein in an amount effective to ameliorate non-specific binding of reagents to the ELISA substrate.

8. A method according to claim 2 or claim 7 wherein said buffer comprises Tris.Cl pH 8.4, sodium lauroylsarcosine, Triton X-100, and bovine serum albumin

(BSA) in the ratio 1 : 3.17 : 3.17 : 3.17.

9. A method according to claim 8 wherein said sample is diluted with said buffer to give a final concentration of 5OmM Tris.Cl pH 8.4, 2.5% w/v sodium lauroylsarcosine, 2.5% v/v Triton X-100, and 2.5% w/v bovine serum albumin (BSA).

10. A method according to claim 6 or claim 7 wherein said ELISA detection is detection via binding to an antibody selected from ICSMlO, ICSMl 8, ICSM33 or ICSM35 antibody or a fragment or fusion thereof.

11. A method according to claim 10 wherein said ELISA detection is detection via binding to ICSMl 0 antibody or a fragment or fusion thereof.

12. Use of ICSM33 antibody or a fragment or fusion thereof in the selective immunoprecipitation of PrPsc.

13. Use of ICSM33 antibody or a fragment or fusion thereof in the ELISA selective detection of PrPsc. ■ •

14. A buffer for the manipulation of PrP, said buffer comprising Tris.Cl pH 8.4, sodium lauroylsarcosine, Triton X-100, and bovine serum albumin (BSA) in the ratio 1 : 3.17 : 3.17 : 3.17. ^•

15. A buffer according to claim 14 wherein said buffer has a concentration of 5OmM Tris.Cl pH 8.4, 2.5% w/v sodium lauroylsarcosine, 2.5% v/v Triton X-100, and 2.5% w/v bovine serum albumin (BSA).

16. Use of a buffer according to claim 14 ' or claim 15 in the immunoprecipitation of PrPsc.

17. Use of a buffer according to claim 14 or claim 15 in the ELISA detection of PrPsc.

18. A method according to any preceding claim wherein said ICSM33 antibody comprises ϊgG₂b mouse immunoglobulin raised against soluble beta-PrP^91"231.

19. A method according to claim 18 wherein said ICSM33 antibody comprises amino acid sequence or comprises CDR amino acid sequence encoded by at least one ICSM33 nucleotide sequence selected from the sequence listing, or a homologue thereof, .

20. A method or use according to any preceding claim wherein said sample comprises blood, or said PrPsc is present in a sample comprising blood.

21. A method of extracting or enriching PrPsc from a sample, said method comprising:

(i) contacting said sample with a reagent which selectively binds PrPsc (ii) incubating said reagent with said sample (iii) separating or concentrating said reagent from said sample wherein said reagent comprises an ICSMlO antibody or a fragment or fusion thereof.

22. Use of ICSMlO antibody or a fragment or fusion thereof in the selective immunoprecipitation of PrPsc.

23. Use of ICSMlO antibody or a fragment or fusion thereof in the ELISA selective detection of PrPsc.

24. A method or use substantially as described herein.

25. A method or use substantially as described herein with reference to the accompanying drawings.