MXPA01003626A - Assay for disease related conformation of a protein - Google Patents

Abstract

An assay method is disclosed which isolates and detects the presence of a disease related conformation of a protein (e.g., PrPSc) present in a sample also containing the non-disease related conformation of the protein (e.g., PrPC). The sample is treated (e.g., contacted with protease) in a manner which hydrolyzes the disease related conformation and not the non-disease related conformation. The treated sample is contacted with a binding partner (e.g., a labeled antibody which binds PrPSc) and the occurrence of binding provides an indication that PrPSc is present. Alternatively the PrPSc of the treated sample is denatured (e.g., contacted with guanadine) or unfolded. The unfolded PrPSc is contacted with a binding partner and the occurrence of binding indicates the presence of PrPSc in the sample. In another embodiment, PrPSc and PrPC are reacted with a labeled antibody that binds both conformations and a conformation that binds only the disease related conformation, and the presence of the disease related conformation is determined by comparing the two.

Description

ESSAY FOR THE CONFORMATION OF A PROTEIN RELATED TO AN ILLNESS Government Rights The Government of the United States may have certain rights in this application pursuant to Law No. AG02132, AG10770, NS22786, NS14069 and NS07219 granted by the National Institute of Health.

Field of the Invention This invention relates to the field of bioassays and more particularly to an assay that makes it possible to isolate and detect a disease conformation of a protein present in a native sample that also contains a non-disease conformation of the protein.

BACKGROUND OF THE INVENTION Prions are infectious pathogens that cause invariably fatal prion diseases (spongiform encephalopathies) of the central nervous system in humans and animals. Prions differ significantly from bacteria, viruses and viroids. The dominant hypothesis is that no nucleic acid is necessary to allow the infection of a prion protein to proceed. A major stage in the study of prions and the diseases they cause was the discovery and purification of a protein designated prion protein [Bolton, McKinley et al. , (1982) Science 218: 1309-131 1: Prusiner, Bolton er al. (1982) Biochemistry 21_: 6942-6950; McKinley, Bolton et al. (1983) Cekk 35: 57-621. Since then, complete genes encoding prion protein have been cloned, ordered in sequence and expressed in transgenic animals. PrPc is encoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell 46: 417-4281 and when PrPc is expressed it is generally found on the outer surface of the neurons. Many lines of evidence indicate that prion diseases result in the transformation of the normal form of the prion protein (PrPc) to the abnormal form (PrPSc). There is no detectable difference in the amino acid sequence of the two forms. However, when the PrPSc is compared to the PrPc it has a conformation with higher β-sheet and lower a-helix content [Pan, Baldwin er al. (1993) Proc Natl Acad Sci USA 90: 1 0962-10966; Safar, Roller er al. (1993) J Biol Chem 268: 20276-202841. The presence of the abnormal form of PrPSc in the brains of humans or infected animals is the only diagnostic marker specific to the disease of prion diseases. PrPSc plays a key role in both the transmission and pathogenesis of prion diseases (spongiform encephalopathies) and is a critical factor in neuronal degeneration [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 103- 143]. The most common prion diseases in animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle rwilesmith and Wells (1 991) Curr Top Microbiol Immunol 172: 21 -381. Four human prion diseases have been identified: (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Streussler-Sheinker disease (GSS) and (4) fatal familial insomnia (FFI). [Gajdusek (1977) Science 197: 943-960; Medori, Tritschler e al al. (1992) N Enal J Med 326: 444-4491. Initially, the presentation of inherited human prion diseases had a conundrum that has since been explained by the cellular genetic origin of PrP. Prions exist in multiple isolates (strains) with different biological characteristics when these different strains infect genetically identical hosts [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 165-186]. The strains differ in incubation time, in PrPSc protein accumulation topology and in some cases also in the distribution and characteristics of cerebral pathology [DeArmond and Prusiner (1997) Greenfield's Neuropathology, 6th Edition: 235-280]. Because PrPSc is the main and most likely the only component of prions, the existence of prion strains has possessed a conundrum regarding how biological information can be encoded in a molecule rather than one comprised of many nucleic acids. It has been found that partial proteolytic treatment of brain homogenates containing certain prion isolates generates peptides with slightly different electrophoretic mobilities [Bessen and Marsh (1992) J Virol 66: 2096-2101; Bessen and Marsh (1992) J Gen Virol 73: 329-334; Telling, Parchi, I went to. (1996) Science 274: 2079-2082]. These findings suggested different sites of proteolytic cleavage due to the different conformation of the PrPSc molecules in the different prion strains. Alternatively, the differences observed could be explained by the formation of different complexes with other molecules, forming different dissociation sites in the different strains of PrPSc [Marsh and Bessen (1994) Phil Trans R Soc Lond B 343: 413-414]. Some researchers have proposed that different prion isolates may differ in the glycosylation patterns of the prion protein [Collinge, Sidle et al. (1996) Nature 383: 685-690; Hill, Zeidler et al, (1997) Lancet 349: 99-100]. However, the reliability of both glycosylation and peptide mapping patterns in the diagnosis of multiple prion strains is currently being debated [Collings, Hill et al. (1997) Nature 386: 564; Somerville, Chong ei al. (1997) Nature 386: 564]. A system for detecting PrPSc by enhancing immunoreactivity after denaturation is provided in Serban, et al. , Neurology, Vol. 40, No. 1, Ja 1990. A direct assay sufficiently sensitive and specific for infectious PrPSc in biological samples could potentially suppress the need for animal inoculations altogether. Unfortunately, such a thing does not seem to be possible with the current PrPSc assays ~ it is estimated that the current sensitivity limit of the proteinase-K and the detection of PrPSc based on Western blot is in the range of 1 μg / ml that corresponds to 104 -105 infectious units of prion. Additionally, the specificity of the traditional assays based on proteinase-K for PrPSc is in question in light of recent findings of only relative or no proteinase K resistance to prion preparations without doubt infectious [ Hsiao, Groth e al. (1994) Proc Natl Acad Sci USA 91: 9126-9130] Telling, e al. (1996) Genes & Dev. Human transthyretin (TTR) is a normal plasma protein composed of four structured units, predominantly β-sheet, identical and serves as a thyroxine hormone transporter. The normal self-assembly of TTR in amyloid fibrils results in two forms of human diseases, namely, systemic senile amyloidosis (PFS) and familial amyloid polyneuropathy (FAP) [Kelly (1996) Curr Opin Strut Biol 6 (1): 1 1 -71 . The origin of amyloid formation in FAP are point mutations in the TTR gene; The origin of the SSA is unknown. The clinical diagnosis is established histologically by detecting amyloid deposits in situ in biopsy material. To date, little is known about the mechanism of conversion of TTR to amyloid in vivo. However, several laboratories have shown that amyloid conversion can be stimulated in vitro by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993) Biochemistry 32 (45): 121 19-27; McCutchen and Kelly (1993) Biochem Biophvs Res Commun 197 (2): 415-21]. The mechanism of the conformational transition involves the monomeric conformational intermediate that is polymerized in structured amyloid fibrils in linear ß sheet [Lai, Colon et al. (1996) Biochemistry 35 (20): 6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxine or triiodophenol [Miroy, Lai et al. (1996) Proc Natl Acad Sci USA 93 (26): 15051 -61. In view of the above, there is clearly a need for a high-transparency, economic assay specifically for examining sample materials for the presence of a pathogenic protein including transthyretin and prion protein.

BRIEF DESCRIPTION OF THE INVENTION There are several known proteins that exist in two or more conformations. Frequently, such proteins have a first conformation which is the normal conformation of the protein and a second conformation related to the disease. Frequently, the two proteins occur together and it is often difficult to determine the presence of conformation related to protein disease because (1) it is difficult to obtain binding partners such as antibodies that bind to this conformation and ( 2) conformation related to the disease is often presented at a relatively low concentration within the sample and relative to the conformation of null disease. The present invention provides test methods utilizing various combinations, enzymes and binding partner treatments such as antibodies for the purpose of detecting disease conformation of the protein. A first test method of the invention comprises the treatment of a sample suspected of containing a protein that involves a first conformation and a second conformation related to the disease with a compound that hydrolyzes the protein in the first conformation but not in the second conformation related to the disease of the protein, thus providing a treated sample. The treated sample is then contacted with a binding partner who joins the conformation related to the disease of the protein. When the linkage is detected, it indicates the presence of the conformation related to the disease of the protein in the sample. The protein detected can be any protein with two or more conformations and is preferably a PrP protein, a TTR protein or an amyloid protein. When the protein is a PrP protein, the compound that hydrolyzes the first conformation of the PrP protein (i.e. PrPc) is Dispase. A second embodiment of the test method of the invention comprises the proportion of a sample suspected of containing a protein that assumes a first conformation and a conformation related to the disease. That sample is then contacted with a compound that hydrolyzes the protein in the first conformation but not in the second conformation related to the disease. This results in a treated sample. The treated sample is then subjected to denaturation. More specifically, the non-hydrolysed protein (if it exists) in the second conformation related to the disease unfolds to a certain degree in order to allow its epitopes to be exposed more fully. The treated, denatured sample is then contacted with a binding partner that binds to the treated, denatured protein, that is, binds to the protein with the exposed epitopes. It is then possible to determine the presence of a protein in the second conformation related to the disease by detecting the linkage of the linking partner.

A third test method of the invention comprises the proportion of a sample suspected of containing a protein that involves a first conformation and a second conformation related to the disease. The sample is contacted with a first binding partner whose binding partner only joins the first conformation of the protein, that is, does not bind to the second conformation related to the disease of the protein. The first binding partner is added in sufficient amounts in order to saturate the sample and bind all and / or substantially all available epitopes on any protein in the first conformation present in the sample. Once the first linking partner has been allowed to join the available epitopes, the sample is then treated with a second linking partner. The second binding partner is different from the first binding partner since the second binding partner joins both the first conformation of the protein and the second conformation related to the disease of the protein. Because all available epitopes in the first conformation protein are busy with the first binding partner, any linkage detected by the second binding partner indicates the presence of the conformation related to the disease of the protein. Depending on the steps used in the assay of the invention, one of two types of antibodies may be used. According to the above, both basic types of sample assay are treated with a compound, for example, a metalloendopeptidase, which selectively hydrolyzes PrPc but not PrPSc. Accordingly, the treated sample can be subjected to two different types of processing, each of which uses a generally different type of antibody. The first general type of antibody selectively binds to the disease conformation of the protein. For example, antibodies that selectively recognize PrPSc bind to an epitope on the C-terminus of the protein. When a PrP protein is in its PrPSc configuration, its C terminus can be linked by antibodies of the type described in the U.S. Patent. 5,846,533 issued December 8, 1998 - reference is also made to WO 98/37210 which claims the description of antibodies that bind to PrPSc. Both publications are incorporated herein by reference in order to describe and expose the antibodies and methods for making antibodies. The second general type of antibody binds to both conformations, of disease and non-disease, of the protein. For example, antibodies that recognize an epitope on the N-terminus of the PrP protein recognize both PrPSc and PrPc after protein denaturation. When the PrP protein is in the PrPSc configuration, the term N is not exposed and as such can not be found by an antibody. To expose an epitope of the term N, PrPSc is denatured, for example, by exposure to guanidine HCl under conditions (pH, temperature and time) which cause the PrPSc to unfold or change its 3-dimensional structure in such a way as to expose a C. terminal epitope C. In this split configuration a wide range of binding partner can be used in detection, including commercially available antibodies, since such antibodies also bind to PrPc, all PrPc must be removed, for example, by selective hydrolysis . An example of an antibody that binds to an epitope of the N-terminus is the monoclonal antibody 3F4 produced by the hybridoma cell line ATCC HB9222 deposited on October 8, 1986 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 and disclosed and described in the US Patent 4,806,627 issued February 21, 1989 - incorporated for reference in order to describe antibody that selectively bind to PrPc in its native form. In addition to the antibody, other binding partners that bind to the conformation unrelated to any disease but not to the conformation related to the disease could be used in the assay of the invention. Antibodies such as 3F4 and others used in the assays described in the examples are commercially available. In one embodiment of the invention, a portion of a sample containing two conformations of a protein (e.g., PrPc and PrPSc) is reacted with a binding partner (e.g., R1) that binds to both conformations and another portion of the sample is reacted with a binding partner (for example, 3F4) that joins only one of the two forms (for example, PrPc). The conformation related to the disease is determined by comparing the two. if the linking partner that joins both conformations shows more linkage than the linking partner that joins only one conformation, this shows that both conformations are presented in the sample. For example, if R1 binds to more protein than 3F4, PrPSc is presented in the sample. No hydrolysis treatment is needed with this method. Nevertheless, the pretreatment can be used and the comparison of the linkage can be adjusted for a variety of factors, for example, binding affinities, comparisons with known samples, hybridization times, signal variations due to secondary antibodies, etc. One aspect of the invention is to provide an immunoassay that is applicable to test samples containing proteins, whose samples are suspected to contain a protein that occurs within a native non-disease conformation and a disease-related conformation (e.g. PrP, ßA4 protein and transthyretin). Another aspect of the invention is to provide an assay that differentiates between (1) the disease-related proteins or portions thereof that are not hydrolyzed by treatment with limited protease, with a protease such as proteinase K (protease-resistant proteins, for example PrP 2730) and (2) disease-related proteins that are hydrolyzed by a limited hydrolyzate treatment with a protease such as proteinase K (e.g., PrPSc sensitive to protease). An advantage of the present invention is that the immunoassay can quickly and accurately determine the presence of the proteins in the disease-related conformation (e.g., PrPSc, ßA4 and transthyretin) even when the antibody used in the assay does not bind or have a low degree of affinity for linkage to the protein in the conformation related to the disease and the conformation related to the disease is present in a concentration lower than that of the non-disease conformation. A feature of the invention is that the obtained signal can be improved by the use of transgenic animals, for example, mice that are used to detect the presence of a protein in a sample. Another feature is that improved fluorescence by dissociation, resolved by time, or a laser-driven, double-wavelength fluorometer, can be used to improve sensitivity. Another advantage is that the assay can detect levels of the disease that causes the conformation of a protein at a concentration of 1 x 103 particles / ml or less. A specific object is to provide a diagnostic assay to determine the presence of infectious prion protein in variable sample materials obtained or derived from tissues and / or body fluids of human, primate, monkey, pig, bovine, sheep, goat, deer, Moose, cat, dog, mouse, chicken and turkey. Another specific object is to provide a diagnostic assay to determine the presence of ßA4 protein in variable sample materials obtained or derived from tissues and / or body fluids of human, primate, monkey, pig, bovine, sheep, goat, deer, elk, cat, dog, mouse, chicken and turkey.

Another object is to provide a rapid assay for the native infectious prion protein in the brains of transgenic and non-transgenic animals injected with sample material that potentially contains prions. Another object is to provide a method for evaluating decontamination procedures by testing the level of denaturation of pathogenic proteins (e.g., prions or β-sheet ßA4) after such treatments. Another advantage is that the process can be carried out without an antibody being directly capable of recognizing an infectious conformation of a protein and without using a proteinase K step to eliminate the signal from normal isoforms (of non-disease) of the protein such as PrPc. Another advantage is that in the invented process there is no need for the antibody to be directly capable of recognizing a pathogenic conformation of ßA4 or transthyretin. An important feature of the assay is the fast, economical and highly transparent design that can be designed with the ability to select 96 samples per day per 96-well plate. Another aspect of the invention is the diagnostic method for quantitatively detecting the TTR in the abnormal amyloid conformation in the sample material obtained from human and animal tissues, body fluids and pharmaceuticals. The invented process provides a direct, sensitive method to distinguish and quantify amyloid and normal conformations of TTR in a mixture present in sample materials. An important object is to provide a specific diagnostic assay for pathogenic TTR in variable sample materials obtained or derived from tissues of human, primate, monkey, pig, bovine, sheep, goat, deer, elk, cat, dog and chicken. Another object is to provide a rapid assay for the amyloid form of TTR in transgenic animals. The specific advantage is that the invented assay can detect pathogenic forms of TTR in a mixture with denatured non-pathogenic forms thereof or in a mixture with a soluble form of TTR - for example, detect less than 1 x 103 particles per ml. These and other objects, advantages and features of the invented process will become apparent to those skilled in the art upon reading the details of the assay method, the development and examination of antibodies and the transgenic mouse as described more fully below in relation to the attached figures.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Before the present assays and methods are discussed and described, it is to be understood that this invention is not limited to antibodies, proteins, labels, assays or particular methods since such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing only particular embodiments and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any method or material similar or equivalent to those described herein can be used in the practice or examination of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in order to set forth and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided for the description only before the filing date of the present application. Nothing herein should be considered as an admission that the present invention is not entitled to antedate such publication by virtue of the prior invention. In addition, the dates of the publications provided are subject to change if the actual publication date is found to be different from the one provided here. DEFINITIONS As used in this, the term "protein" is intended to encompass any amino acid sequence and includes modified sequences such as glycoproteins. The term includes naturally occurring proteins and peptides as well as those that are synthesized recombinantly or synthetically. As used in connection with the present invention, the term "protein" is specifically proposed to cover naturally occurring proteins that appear in at least two different conformations where both conformations have the same or substantially the same amino acid sequence but have different structures three-dimensional The two conformations of the protein include at least one conformation that is not related to a disease state and at least one conformation that is related to a disease-pathogenic state. A specific and preferred example of a protein, as used in connection with this disclosure, is a PrP protein that includes the non-disease form referred to as the PrPc form and the disease-related form referred to as the PrPSc. Although a prion protein or the PrPSc form of a PrP protein is infectious and pathogenic, the conformation of the disease of other proteins is not infectious but is pathogenic. As used herein, the term "pathogenic" may mean that the protein actually causes the disease or may simply mean that the protein is associated with the disease and therefore occurs when the disease is present. Thus, a pathogenic protein, as used in connection with this exposure, is not necessarily a protein that is the specific causative agent of a disease. The terms "pretreatment", "splitting treatment" and "limited protease treatment", attempt to encompass the descriptions and uses of these terms as provided in the respective sections having these titles. The terms "PrP protein", "PrP" and the like are used interchangeably herein and mean both the infectious particle form PrPSc known to cause diseases (spongiform encephalopathies) in humans and animals as well as the non-infectious form PrPc which, under appropriate conditions, is converted to the infectious form PrPSc. The terms "prion", "prion protein" and "PrPSc protein" and the similar ones used interchangeably herein refer to the PrPSc form of infectious PrP and are a contraction of the words "protein" and "infection" " The particles are largely, if not exclusively, comprised of PrPSc molecules encoded by a PrP gene. Prions are different from bacteria, viruses and viroids. The known prions infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of goats and sheep, as well as bovine spongiform encephalopathy (BSE) or "mad cow disease" and feline spongiform encephalopathy of cats. Four prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Straussler-Scheinker disease (GSS) and (4) fatal familial insomnia ( FFI). As used herein, "prion" includes all forms of prions that cause all or any of these diseases or others in any animal used - and in particular in humans and domesticated farm animals. The term "PrP gene" is used herein to describe genetic material that expresses proteins that include known polymorphisms and pathogenic mutations. The term "PrP gene" generally refers to any gene of any species that encodes any form of a PrP protein. Some commonly known PrP sequences are described in Gabriel et al. , Proc.Natl. Acad. Sci. USA 89: 9097-9101 (1992) and the US Patents. 5,565, 186; 5,763,740; 5,792,901 and WO 97/04814, incorporated herein by reference in order to set forth and describe such sequences. The PrP gene can be from any animal, including the "host" and "test" animals described herein and any and all polymorphisms and mutations thereof, recognizing that the terms include such other genes that are yet to be discovered. The protein expressed by such a gene can assume either a form of PrPc (of no disease) or of PrPSc (of disease). The term "binding partner" refers to any molecule that binds to the target molecule of interest. Preferably, the binding is sufficiently high affinity to make it possible to link to target molecules of interest present at a low concentration, for example, 1 x 103 particles per ml or less. More preferably, the binding partner is selective in the binding of only the target molecule and not other molecules. The preferred binding partners are antibodies as defined below. The term "antibody" refers to an immunoglobulin protein that is capable of binding to an antigen. As used herein, "antibody" is meant to include the total antibody, as well as any antibody fragment (eg, F (ab) ', Fab, Fv) capable of binding to the epitope, antigen or antigenic fragment of interest. Antibodies for assays of the invention can be immunoreactive or immunospecific and therefore specifically and selectively bound to a protein of interest, for example, an A4β amyloid protein or a PrP protein. Antibodies that are immunoreactive and immunospecific for both the native non-disease form and the disease form treated but not for the untreated form of disease, (e.g., both for native PrPc and for PrPSc treated but not for non-native PrPSc ) can be used because the sample is treated to remove, ie, hydrolyze, PrPc. Antibodies to PrP are preferably immunospecific - for example, not substantially trans-reactive with related materials. Some specific antibodies that may be used in connection with the invention are described in the published PCT application WO 97/10505, which is incorporated herein by reference in order to set forth and describe antibodies. This published PCT application corresponds to USSN 08 / 713,939. The antibodies set forth in the PCT application that bind to PrPSc can be used to carry out the basic assay of the present invention when the sample has been treated with enough dispase to hydrolyse all or substantially all of the PrPc present in the sample. Another antibody useful for binding to PrPc is the monoclonal antibody 263K 3F4 produced by the hybridoma cell line ATCC HB9222 deposited on October 8, 1986 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 and exposed and described in the U.S. Patent 4,806,627 issued February 21, 1989 - incorporated for reference in order to expose the antibodies that bind selectively to PrPc. The term "antibody" encompasses all types of antibodies, for example, polyclonal, monoclonal, and those produced by the phage display methodology. Particularly preferred antibodies of the invention are antibodies that have a relatively high degree of affinity for both native PrPc and treated PrPSc but a relatively low or substantially zero degree of affinity for PrPSc. More specifically, the antibodies of the invention preferably have four times or more, more preferably fifteen times or more, and even more preferably 30 times or more binding affinity for both native PrPc and denatured PrPSc compared to binding affinity to the Native PrPSc. "Purified antibody" refers to one that is sufficiently free of other proteins, carbohydrates and lipids with which it is naturally associated. Such an antibody "preferentially binds" to a conformation of denatured disease of a protein such as the β-sheet conformation of A4β or PrPSc protein (or an antigenic fragment thereof) and does not recognize or substantially bind to other antigenically non-antigenic molecules. related A purified antibody of the invention is preferably immunoreactive and immunospecific for a specific species and more preferably immunospecific for the native PrPc and for denatured forms of PrPc and PrPSc or, alternatively, for native or untreated PrPSc. "Antigenic fragment" of a protein (e.g., a PrP protein) means a portion of such a protein that is capable of binding to an antibody.

By "specifically binding" is meant a linkage of high avidity and / or high affinity of an antibody to a specific polypeptide, for example, epitope of a protein, for example, denatured PrPSc or denatured A4β protein. The binding of the antibody to its epitope on this specific polypeptide is preferably stronger than the binding of the same antibody to any other epitope, particularly those that may occur in molecules in association with, or in the same sample as, the specific polypeptide of interest, for example, it binds more strongly to the epitope fragments of a protein such as PrPSc so that when adjusting binding conditions the antibody binds almost exclusively to an epitope site or fragments of a desired protein, such as a fragment. of epitope exposed by denaturation of PrPSc and not exposed to native PrPSc. By "detectably labeled antibody", "detectably labeled anti-PrP" or "detectably labeled anti-PrP fragment" is meant an antibody (or antibody fragment that retains binding specificity) having an attached detectable label. The detectable label is normally attached by chemical conjugation, but when the label is a polypeptide, it could be alternatively annexed by genetic design techniques. Methods for the production of detectably labeled proteins are well known in the art. Detectable labels known in the art are usually radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase) or other elements or compounds that either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emitted a detectable signal after exposure of the label to its substrate. Various pairs of detectable label / substrate (eg, horseradish peroxidase / diaminobenzidine, avidin / streptavidin, luciferase / luciferin), methods for labeling antibodies and methods for using labeled antibodies are well known in the art (see, for example, Harlow and Lane, eds (Antibodies: A Laboratory Manual (1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) Europium is a particularly preferred label The abbreviations used herein include: CNS for the central nervous system; BSE for bovine spongiform encephalopathy, CJD for Creutzfeldt-Jacob disease, FFI for fatal familial insomnia, GnnHCI straw Guanidine hydrochloride, GSS for Gerstamnn-Strassler-Scheinker disease, Hu for human, HuPrP for human prion protein, Mo for mouse; MoPrP for prion protein for mouse; SHa for a Syrian hamster; SHaPrP for Syrian hamster prion protein; Tg for transgenic; Tg (SHaPrP) for a transgenic mouse containing the PrP gene of a Syrian hamster; Tg (HuPrP) for transgenic mice containing the complete human PrP gene; Tg (ShePrP) for transgenic mice that contain the complete goat PrP gene; Tg (BovPrP) for transgenic mice that contain the full cow PrP gene; PrPSc for the scrapie isoform of prion protein; PrPc for the cellular isoform, normal, common, contained in the prion protein; PrP 27-30 or PrPSc 27-30 for the treatment or protease-resistant form of PrPSc, MoPrPSc for the scrapie isoform of the mouse prion protein; MHu2M for a mouse / human chimeric PrP gene wherein a region of the mouse PrP gene is replaced by a corresponding human sequence that differs from the mouse PrP at 9 codons; Tg mice (MHu2M) are transgenic mice of the invention that include the chimeric gene MHu2M; MHu2MPrPSc for the scrapie isoform of the chimeric gene of PrP of human / mouse; PrPC D for the CJD isoform of a PrP protein; Prnp0 / 0 for the ablation of both alleles of an endogenous prion protein gene, for example, the MoPrP gene; Tg (SHaPrP + 0) 81 / Prnp0 0 for a particular line (81) of transgenic mice expressing SHaPrP, +/- 0 indicates heterozygosity; Tg (HuPrP) / Prnp0 0 for a hybrid mouse obtained by crossing a mouse with a human prion protein gene (HuPrP with a mouse with both alleles of the fractionated endogenous prion protein gene; Tg (MHu2M) / Prnp ° '° for a hybrid mouse obtained by cross-linking a mouse with a chimeric prion protein gene (MHu2M) with a mouse with both alleles of the fractionated endogenous prion protein gene, TTR for transthyretin, FVB for a consanguineous strain of mice frequently used in the production of transgenic mice since the eggs of FVB mice are relatively large and tolerate the microinjection of exogenous DNA relatively well; [PrPβ] - concentration of prion protein in ß sheet conformation; [ßA4β] - concentration of ßA4 in sheet conformation ß; [DRC] - concentration of a conformation related to the disease of a protein GENERAL ASPECTS OF THE INVENTION The test method the proportion of a sample suspected of containing a protein that assumes a first conformation and a second conformation related to a disease and is capable of detecting a disease conformation of the protein when it occurs at a very low concentration in relation to the concentration of other proteins and compounds that include the conformation of no disease. The exposed test methods allow one to isolate and detect the presence of a conformation related to a disease of a protein (e.g., PrPSc) present in a sample that also contains the conformation unrelated to any disease of the protein (e.g. PrPc). The sample is treated (eg, dispase is contacted) in a manner that hydrolyzes the PrPc and not the PrPSc. The hydrolyzing reaction is stopped (for example, by the addition of EDTA). The treated sample is contacted with a binding partner (eg, a labeled antibody that binds PrPSc) and the occurrence of linkage provides an indication that PrPSc is present. Alternatively, the PrPSc. of the treated sample is denatured (eg, it is contacted with guanidine) or unfolded so that it has exposed epitopes. The split PrPSc is contacted with a binding partner (for example, 3F4 tagging) and the occurrence of linking indicates the presence of PrPSc in the sample. In accordance with any of the test modalities, it is preferable to pre-treat the sample being examined to (1) remove as much contaminating protein as possible; and (2) increasing the concentration of the protein related to the disease in the sample relative to the conformation unrelated to any disease of the protein. For example, the initial sample can be chemically treated with a compound that preferentially degrades or denatures the contaminating proteins and / or the relaxed non-diseased form of the protein and / or is expressed for antibodies that preferentially bind to (in order to remove) contaminants and / or the conformation of no protein disease. It may be possible to further improve the sensitivity of various aspects of the invention by concentrating the conformation of the disease of a protein by the addition of a compound that selectively binds to the conformation of the disease in order to form a complex and centrifuge the sample to separate by precipitation the complex that is then examined according to the methods described herein. The specificity with respect to such concentration methods is described in detail in our co-pending application Serial No. 09 / 026,967 entitled "Process for Concentrating Disease-Related Protein with Conformation". The different embodiments of the assay of the invention described above are all "direct" types of immunoassays - it means that the sample is tested directly with the antibody labeled either with or without treatment to change the conformation of any conformation protein related to the disease, present in the sample. An "indirect" trial could also be used. For example, it may be desirable to improve the number of proteins related to the disease in the sample (if they exist). by using a transgenic mouse and thus improve any signal obtained. To carry out these embodiments of the invention, the sample is first used to inoculate a transgenic mouse that has modified its genome in order to develop disease symptoms when it is inoculated with proteins in the conformation related to the disease. After the mice are inoculated, a sufficient period of time is allowed (for example, 30 days) after which the transgenic animal is sacrificed and a sample, such as a homogenized brain tissue, is used in the direct assay described above. The present invention improves the ability of transgenic mice to detect prions by shortening the period of time that must pass until a determination can be made as to whether the original sample includes proteins in the conformation related to the disease. It would also be possible to use mice of the type disclosed and described in any of the US Patents. 5,565, 186; 5,763,740; or 5,792,901 or the application of a PrP directed to epitope as set forth in the U.S. Patent. 5,750,361 to affinity purify the PrPSc from the brain of a Tg mouse and, therefore, apply the assay of the present invention. Without the present invention, the mouse is inoculated and must be waited until the inoculated mouse actually demonstrates symptoms of the disease. Depending on the mouse, this can take several months or even years. Any of the assays of the present invention could be used with any transgenic mouse such as those described above. The assay could be used well before the mouse develops symptoms of disease, thus shortening the time needed to determine whether a sample includes infectious proteins. The assay methodology of the present invention can be applied to any type of sample when it is suspected that the sample contains a protein that occurs in at least two conformations. The protein must occur in a conformation that binds to known antibodies, antibodies that can be generated or other specific binding partners. The second conformation must be sufficiently different from the first conformation in terms of its ability to hydrolyze by the compound (eg, dispase). In its conceptually simpler form, the invention works best when a compound rapidly and completely hydrolyzes the non-diseased conformation of the protein without affecting the conformation related to the disease. However, in reality, a given protein has more than two conformations. The protein may have more than one non-disease conformation and more than one conformation related to the disease, (Telling, et al., Science (1996)). The invention is still useful when there are multiple conformations of non-disease and disease forms of the protein ~ taking into account that (1) at least one non-disease conformation differs from at least one disease conformation in terms of its ability to hydrolyse by a compound As indicated above, the assay of the invention can be used to test any type of sample for any type of protein, taking into account that the protein includes a non-disease conformation and one related to the disease. However, the invention was developed particularly to test samples for the presence of (1) PrP proteins and to determine whether the sample included a PrP protein in its disease conformation, ie, it included PrPSc (2) insoluble forms of βA4 associated with Alzheimer's disease and (3) transthyretin. Accordingly, most of the following discussion is directed to the use of the immunoassay of the present invention to detect the presence of any PrPSc (or to a lesser extent ßA4 or transthyretin (TTR) in a sample - it being understood that the same general concepts are applicable to detect conformations related to the disease from a wide range of different types of proteins). The europium labeled antibodies used (3F4) have a high binding affinity to PrPc (non-disease conformation) comprising a rich a-helical conformation. The antibodies have a low binding affinity with PrPSc (conformation of the disease) comprising a conformation rich in ß sheet. IgG can be obtained from common, monoclonal, polyclonal or recombinant antibodies, which typically recognize the 90-145 sequence of PrPc and the conformationally cleaved prion protein. The different conformations of recombinant prion protein. HE. chemically degraded polystyrene plates through a glutaraldehyde activation stage. The relative affinities of Eu-labeled IgG with an α-helical, ß- and helical-random conformation of recombinant Syrian hamster prion protein, corresponding to the sequence 90-231, were determined by improved fluorescence by dissociation, resolved by time in a 96-well polystyrene plate format. After the labeled antibodies have been provided with sufficient time, temperature and chemical conditions (e.g., pH) to bind to the appropriate proteins present in the respective portions, the level of binding of the labeled antibody to the protein is determined. Once a labeled antibody has been bound to its target, detection can be difficult due to the low concentration of the target molecule in the sample. For detection, different procedures can be used. Improved fluorescence by dissociation, resolved by time and more preferably laser-driven, double-wavelength fluorometers, are particularly useful devices - see Hemmilá et al. , Bioanalytical Applications of Labeling Technologies (eds Hemmilá) 1 13-1 19 (Wallas Oy. Turku, Finland, 1995). These devices make it possible to detect concentrations in an amount in the range of about 1 x 103 particles per ml or less. A high degree of sensitivity is preferred because in most samples the concentration of protein in the conformation of the disease will be very low. For example, the non-diseased conformation of the protein could be present in an amount of about 1 x 108 particles / ml while the disease conformation of the protein only occurs in an amount of 1 x 104 particles / ml. The assay can be used to examine the presence of the disease conformation of a given protein within any type of sample. Some of the most typical samples to be examined include pharmaceuticals that include components that are derived from living mammals or use materials derived from living mammals in their processing. It would also be desirable to examine transplant organs and food items such as those suspected of containing infectious prions. The invention could be used to examine the presence of the disease conformation of one or more types of proteins such as infectious PrPSc in pharmaceuticals, cosmetics, biopsy or autopsy tissue, brain, spinal cord, peripheral nerve, muscle, cerebrospinal fluid, blood and blood components, lymph nodes and in cultures derived from humans or animals, infected or potentially infected by disease forms of proteins such as prions. The brains of cows suspected of being infected with prions (ie, BoPrPSc) could be examined to determine if cows can be safely used for human consumption. GENERAL TREATMENT An assay of the invention can use all or any of the three basic types of treatment defined above. The treatments are (1) pretreatment, (2) splitting treatment and (3) hydrolysis treatment. In general, the conditions for the pretreatment are mild, those for the treatment of moderate splitting and those for the treatment of hard hydrolysis. Each type of treatment can use the same means (for example, proteases, time, pH, temperature, etc.) but each uses a different degree, for example, higher concentration, longer time, higher temperature. However, the hydrolysis treatment should employ a compound that selectively hydrolyzes only the non-disease conformation and not the conformation of the disease.

PRETREATMENT Before carrying out the treatment or examination of antibodies in the sample, it may be desirable to subject the sample to pretreatment. The pretreatment is carried out in order to destroy or remove the unrelated proteins as well as some form of non-disease of the protein present within the sample. Examples of pretreatment methodology include the production of a column that includes antibodies bound to support surfaces that the antibodies bind to the non-diseased conformation of the protein, thereby removing as much as possible of the non-diseased conformation of the proteins. Antibodies that bind to common but unrelated protein can also be used. Alternatively, the sample can be subjected to physical treatment such as long-term hydrostatic pressure or temperature alone or in combination with chemicals such as acids or alkalis, as indicated above to destroy proteins present in the sample whose proteins are not related to those that are tested or are in the conformation of no disease. In some cases, the proteins in the non-disease and disease conformation will be destroyed. However, a greater relative percentage of the proteins in the non-disease conformation will be destroyed because these proteins are initially in a looser conformation that is more vulnerable to destruction. Thus, the pretreatment methodology results in a sample that includes a relatively lower concentration of the non-diseased conformation of the protein relative to the concentration of the disease conformation of the protein. In addition, the pretreated sample will have a lower concentration of unrelated proteins. This increases the sensitivity of the assay, making it possible to detect lower concentrations of the disease's conformation of the protein. The removal of proteins is preferred over the destruction of such, since the destruction will decrease the sensitivity if the conformation of the disease is destroyed. A particularly useful pretreatment method is set forth in our patent application Serial No. 09 / 023,967 entitled "Process for Concentrating Disease-Related Protein with Conformation". BREAKING TREATMENT The splitting treatment denatures the protein but does not hydrolyze the proteins of interest and may include the exposure of the proteins to any physical and / or chemical medium that results in the protein that is originally present in a conformation related to the disease adjusted (eg, PrPSc) assume a more relaxed conformation that has a higher degree of binding affinity for any binding partner, such as antibodies (eg, exposing an epitope of terminal N of PrPSc). In general, the cleavage treatment involves securing the protein to some media that originate epitopes in the protein that were not previously exposed or partially exposed to expose or become more exposed so that an antibody or other binding partner can bind more easily to the recently exposed epitope. The methods used for the splitting treatment may include: (1) physical, such as hydrostatic pressure or temperature, (2) chemical, such as acidic or alkaline pH, chaotropic salts, denaturing detergents, guanidine hydrochloride and proteinases such as Proteinase K and (3) combinations of the above. The concentration, temperature and time should be considered in order to obtain the desired effect, for example, cleavage but not hydrolysis. The treatment time will vary depending on the treatment used, but it must be carried out for a sufficient time to obtain the desired effect, for example, splitting treatment to expose new binding sites but not so long as to completely denature or hydrolyze the protein. When the PrP protein unfolding treatment is carried out without chemical treatment, the temperature is raised to about 40 ° C to about 80 ° C for a sufficient time to obtain the desired amount of PrPSc cleavage. The temperature may be lower and the time may be shorter if the pH rises to 12 or 13. HYDROLYSIS TREATMENT The hydrolysis treatment is a UTI treatment which is the most important treatment method used in a test modality of the invention. After a sample has been subjected to the pretreatment treatment, it is subjected to the hydrolysis treatment. This treatment will destroy or hydrolyse all or substantially all of the protein in the sample that is in the non-disease conformation and will not hydrolyze the protein in the conformation of the disease. The hydrolysis treatment is preferably carried out by an enzyme such as a hydrolase acting on the peptide bonds, preferably a neutral protease, more preferably a metalloendopeptidase and more preferably a leucostome peptidase or peptidase A. The proteases used in the method of the invention can be used alone, in combination or in conjunction with enzymes having similar but distinct activity, such as carbohydrase, for example, collagenase, amylase or serine alkaline protease. The concentration of the treatment compounds as well as the time and temperature will vary with the protein to be treated and the final result to be obtained. For example, with PrP the treatment is carried out to hydrolyze all or substantially all of the non-PrPc present, but does not hydrolyze the PrPSc present. The object of this treatment is to hydrolyze as much non-disease protein as possible (preferably whole) while hydrolyzing as little protein (preferably nothing) related to the disease as possible. The treatment is preferably designed in such a way that it can be stopped quickly and completely at any given time. For example, hydrolysis of PrPC with dispasses or other related proteases can be stopped by the addition of EDTA. The following list of enzymes are the preferred compounds of the method of the invention: The method of the invention is not limited to these enzymes, and therefore other enzymes predicted by those skilled in the art can be used to function in the method of the invention. LINKAGE PROTEINS TO SUPPORT SURFACES The method of chemical or affinity coupling of PrP protein to the plastic support is generally described in the available literature and may vary. The antibodies used in the diagnostic assay are polyclonal, monoclonal or recombinant Fab and need to be specific species with preferential binding to the native PrPc or denatured form of PrPSc with a reactivity at least preferably 4 < go less times with the infectious PrP, assuming the same amount of the antigen. USING THE PRISM DETECTION TEST (PrPSc) One aspect of the invention is a two-step process for diagnosing prion disease by quantitatively measuring the native infectious form of the PrPSc protein in the sample material or in the brains of susceptible animals inoculated with such material. The sample is preferably pretreated to remove as much non-disease protein and not related to any disease as possible. The pretreated sample is first subjected to hydrolysis treatment and then degraded to the plastic support.

To measure the concentration of PrPSc when it is much lower than PrPc, the detection system must have an extreme sensitivity and a linear range of at least 104. The assay described here can easily detect PrPSc at a concentration of (approximately) 50 pg / ml by the use of IgG labeled with europium. Assuming 105-106 molecules of PrPSc per unit ID50, the present assay can easily detect 5 x 102-5 x 103 ID50 units per ml. The assay can detect PrPSc in mixtures (by direct method) where the PrPSc concentration is less than 1% of the PrPc concentration. Additional sensitivity can be achieved by immunoprecipitation, using a sandwich format for a solid state assay, differential centrifugation with detergent extraction to remove PrPc, the indirect method of transgenic animal or combinations of these methods. A conservative estimate is that such procedures should allow measurement of between 5 and 50 ID50 units per ml or less conservatively to measure between 0.1 and 0.01 ID50 units per ml. Such measurements would provide a quick, "positive" means to establish biological sterility, which is the absence of infectivity. ANTIBODIES The method for generating antibodies is generally known to those skilled in the art. Since the disease form is often found in a tighter configuration than the non-disease form, with fewer epitopes exposed, one can easily generate antibodies that bind only to the non-disease form of the protein or the form of disease treated. For example, antibodies that detect treated forms of PrPSc protein and PrPc protein can be generated by immunizing rabbits or mice with a-helical conformations of recombinant PrP, native PrPc from animal brains, synthetic peptides in helical or helical conformations. randomized, or against denatured PrPSc or PrP 27-30. Only antibodies with affinity at least 4 times greater than PrPc (or the denatured conformation of PrPSc from the same species) should be selected in comparison with their affinity to PrPSc. The method of antibody generation, purification, labeling and detection may vary. The IgG or Fab can be purified from different sources by affinity HPLC using a Protein column A and size exclusion HPLC. The purified antibodies can be labeled with Europium and detected by time resolved fluorescence. The binding of the antibody to different PrP protein linkages can be measured by fluorescence enhanced by dissociation, resolved by time. However, the detection system of IgG bound to PrP on a solid support in situ or in solution may vary. In addition, it is possible to use direct or indirect immunological methods that include direct radiolabels, fluorescence, luminescence, avidin-biotin amplification or enzyme-linked assays with luminescent or colored substrates. An antibody that can be used in the invention is disclosed in US 4,806,627, issued February 21, 1989, which discloses the monoclonal antibody 263K 3F4, produced by the ATCC cell line HB9222 deposited on October 8, 1986, which is incorporated in the present for reference. The cell line that produces the antibody can be obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852. In general, scrapie infection fails to produce an immune response, host organisms being tolerant to the PrPSc of the same species. Antibodies that bind to either PrPc or PrPSc are disclosed in the U.S. Patent. 5,846,533 issued December 8, 1998. Any antibody that binds PrPc and not PrPSc can be used, and those skilled in the art can generate them by using known procedures, for example, see methods for producing antibody libraries of roll-out per page in EU 5,223,409. It has been considered to raise polyclonal anti-PrP antibodies in rabbits after immunization with large amounts of formic acid or SHaPrP 27-30 denatured by SDS [Bendheim, Barry et al. , (1984) Nature 310: 418-421; Bode, Pocchiari eí al. (1985) J Gen Virol 66: 2471-2478; Safar, Ceroni e al. (1990) Neurology 40: 513-517]. Similarly, a handful of anti-PrP monoclonal antibodies against PrP 27-30 has been produced in mice [Barry and Prusiner (1986) J Infect Dis 154: 518-521; Kascsak, Rubenstein eí al. , (1987) J Virol 61: 3688-3693]. These antibodies were generated against PrP 27-30 denatured with formic acid or SDS and are capable of recognizing native PrPc and treating or denaturing PrPSc from both SHa and humans equally well, but not from binding to native PrPSc. Not surprisingly, the epitopes of these antibodies were plotted for regions of the sequence containing amino acid differences between SHa and MoPrP [Rogers, Yehiely et al. (1993) Proc Natl Acad Sci USA 90: 3182-3186]. It is not entirely clear why many of the antibodies of the type described in the above publications will bind PrPc and PrPSc treated or denatured but not native PrPSc. Without being related to any particular theory, it is suggested that such a situation takes place because the epitopes that are exposed when the protein is in the conformation of PrPc are not exposed or are partially hidden in the PrPSc configuration - where the protein is relatively insoluble and folded together more compactly. For purposes of the invention, an indication that no linkage occurs means that the affinity constant or equilibrium Ka is 106 I / mol or less. In addition, the linkage will be recognized as existing when the Ka is 107 I / mol or greater, preferably 108 l / mol or more. The binding affinity of 107 I / mol or greater may be due (1) to a single monoclonal antibody (ie, large numbers of one type of antibody) or (2) to a plurality of different monoclonal antibodies (e.g., large numbers of each of five different monoclonal antibodies) or (3) large numbers of polyclonal antibodies. It is also possible to use combinations of (1) - (3). The selected preferred antibodies will bind at least 4 folds more avidly to the treated or denatured forms of PrPSc of the protein compared to its binding to the native conformation of PrPSc. The four-fold differential in binding affinity can be carried out by using several different antibodies according to (1) - (3) above and as such some of the antibodies in a mixture could have a difference of less than 4 folds. A variety of different types of assays of the invention can be used with one or more different antibodies. Those skilled in the art will recognize that antibodies can be labeled with known labels and used with currently available robotics, sandwich assays, electronic detectors, flow cytometry and the like. DISEASES ASSOCIATED WITH INSOLUBLE PROTEINS Much of the discussion and the specific examples provided here relate to the use of the assay in connection with the determination of the presence of PrPSc in the sample. However, as indicated above, the assay of the invention can be applied in determining the presence of any protein that assumes two different conformational forms, one of which is associated with the disease. The following is a non-limiting list of diseases with associated insoluble proteins that assume two or more different conformations. Disease Insoluble Proteins Alzheimer's Disease APP, peptide Aß, a1 - antichymotrypsin, tan, component without Aß Diseases of prion, Creutzfeld disease Jakob, scrapie and prpsc spongiform encephalopathy ALS SOD and neurofilament Pick disease Pick body Parkinson's disease Lewy body Type 1 Diabetes Amilin Multiple myeloma - plasma cell dyscoria IgGT chain Familial amyloidotic polyneuropathy Transthyretin Spinal cord carcinoma Thyroid Procalcitonin Chronic renal failure ß - microglobulin Congestive heart failure Atrial natriuretic factor Systemic and senile cardiac amyloidosis Transthyretin Chronic inflammation Amyloid serum A Atherosclerosis ApoA1 Familial amyloidosis Gelsolin It should be noted that the insoluble proteins listed above each include a number of variants or mutations that give as a result, different strains are all encompassed by the present invention. The mutations and pathogenic polymorphisms known in the PrP gene related to the above diseases are given below and the human sequences, sheep and cattle are given in EU 5,565, 1 86, issued on October 15, 1996.

MUTATION TABLE Human Mutations Polymorphisms Polymorphisms Human Pathogenic Polymorphisms of Sheep Cattle Insertion of 2 Codon 129 Codon 171 5 or 6 octarepeats Met / Val Arg / Glu octarepeats Insertion of 4 Codon 219 Codon 136 octarepeats Glu / Lys Ala / Val Insertion of 5 octarepetences Insertion of 6 octarepetences Insertion of 7 octarepetences Insertion of 8 octarepetences Insertion of 9 octarepetitions Codon 102 Pro-Leu Codon 105 Pro-Leu Codon 1 17 Ala-Val Detention Codon 145 Codon 178 Asp-Asn Codon 180 Val-lie Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210 Val-lie Codon 217 Asn - Arg Codon 232 Met- Ala It should be noted that such proteins have two different 3-dimensional conformations with the same amino acid sequence. One conformation is associated with the characteristics of the disease and is generally insoluble while the other conformation is not associated with the characteristics of the disease and is soluble. The methodology of the present invention is not limited to the diseases, proteins and strains listed. DETECTION OF THE SHEET FORM ß FROM ßA4 One aspect of the invention involves a two-step process for diagnosing Alzheimer's disease based on the presence of a constricted form of a protein (ßA4 amyloidosis) by quantitative measurement of the leaf shape ß of the ßA4 protein in the sample material, for example, in the brain or body fluids. The sample is divided into two aliquots. The first aliquot is degraded on a solid plastic support (long chain polymeric material) in the native conformation through a chemical activation step under non-denaturing conditions. The second portion of the sample is first subjected to splitting treatment and then degraded into a plastic support. Both portions of the sample material react in situ with the labeled antibodies that preferentially recognize soluble ßA4 or ßA4 from cleavage treatment of human or a given animal species. The amount of the antibody bound to unfolded or native conformations of the βA4 protein is recorded by the labeled secondary antibody signal. The excess of the signal obtained with the sample treated by splitting compared to the expected change in the signal obtained with the native a-helical conformation of the ßA4 protein is the measurement of the amount of ßA4 structured in ß sheet in the original sample . The formula developed for calculating the content of ßA4 is given above in connection with the calculation of PrPSc content. The diagnosis of amyloidosis of ßA4 (Alzheimer's disease) is established by three procedures: (1) measurement of the denatured sample alone and by detecting the increase in the total amount of ßA4 (concentration) in the sample examined above the levels previous soluble ßA4, obtained from normal controls; (2) calculation of the ratio between treated by splitting versus native signal for a given antibody (protein index) - for example, values greater than 2 of the monoclonal antibody 6F3D and the secondary antibody labeled with europium; (3) the evaluation of the change of the denatured sample signal on the expected change in the signal for the α-helical conformation of ßA4 as a measure of the amount of ßA4 structured in infectious ß sheet in the original sample. The formula developed for calculating the content of ßA4 is given above.

The particular strain of ßA4 can also be determined by using the same methodology described above to determine the strain of PrPSc in a sample. The invention provides a direct diagnostic method for detecting the presence of pathogenic forms of βA4 protein in pharmaceuticals, biopsy or autopsy tissue, brain, spinal cord, peripheral nerves, muscle, cerebrospinal fluid, blood and blood components, lymph nodes and in cultures derivatives of animals or humans that express or potentially express ßA4 protein. The invention also makes it possible to follow the conformational transition of α-helical-to-sheet-ß of the ßA4 protein, or its fragments of synthetic or recombinant origin, and to provide a method for selecting compounds for their ability to stabilize the normal soluble conformation of the ßA4 protein and thus prevent conversion to ßA4 protein structured in ß sheet and insoluble, pathogenic. Typical methods of denaturation of the sample include: (1) physical, such as pressure or hydrostatic temperature, (2) chemical, such as acidic or alkaline pH, chaotropic salts or denaturing detergents, and (3) combination of the above. Chemical or affinity coupling methods of ßA4 protein to a plastic support are described in the available literature and may vary. The antibodies used in the diagnostic assay can be polyclonal, monoclonal or recombinant Fab and must be specific species with preferential linkage to the soluble or denatured form of ßA4 with preferably at least a 2 fold difference in reactivity between ßA4 a-helical and structured with ß sheet, assuming the same amount of antigen. The methods of securing the sample to the plastic support may vary and may be covalent or non-covalent, as described in the available literature. The sensitivity of the assay described in the examples can be increased by the use of high affinity antibodies, sandwich format, immunoprecipitation or differential centrifugation. However, only antibodies with an affinity of at least 2 folds for the doubling treatment compared with the ßA4 native leaf conformation of the same species should be used for the diagnostic assay. The methods of antibody generation, purification, labeling and detection may vary. The binding of antibodies to different conformations of ßA4 protein was measured by fluorescence enhanced by dissociation, resolved by time. However, the IgG detection system bound to ßA4 on solid support in situ or in solution may vary and may use direct or indirect immunological methods that include direct radiolabels, fluorescence, luminescence, avidin-biotin amplification or enzyme-linked assays. luminescent or colored substrates. EXAMPLES The following examples are set forth in order to provide those skilled in the art with a full disclosure and description of the manner of making and using the assays of the present invention and are not intended to limit the scope of what the inventors consider to be their invention, nor do they attempt to represent or imply that the following experiments are all nor the only experiments carried out. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.) but some errors and experimental deviations must be taken into account. Unless indicated otherwise, the parts are parts by weight, the molecular weight is average molecular weight, the temperature is in degrees centigrade and the pressure is almost or is atmospheric. EXAMPLE 1 DETECTION OF PrPSc IN HAMSTER BRAIN To determine the levels of PrPSc in affected hamsters, a prion-infected hamster and a normal hamster were each sacrificed and their brains removed. A 10% homogenate (w / v) of each of the brains was prepared by dispersing brain tissue in PBS. The brain homogenate was then subjected to a low speed centrifugation of 500xg for 15 minutes to separate the suspended proteins from the unwanted cell debris. The total protein concentration of the supernatant (SI) was measured by the use of a BCA Protein Assay (Pierce) and the concentration of each brain homogenate was adjusted with PBS to 3.5 mg / ml. A portion of the homogenate was saved to serve as a control of the total brain proteins. Metalloendopeptidase (Worthington) dispase was added to the rest of each sample at an enzyme to protein ratio of 1: 35. The homogenates were digested with 100 μg / ml dispase for 60 minutes at 37 ° C in the presence of either 0.1 5% Zwittergent 3-1 2 (Calbiochem) or 0.2% or 2% sodium dodecyl sarcosinate (Sarkosyl ). A sample was also placed without the dispase at 37 ° C to serve as a digestion control. After digestion, the dispase is inactivated by the addition of 50 mM EDTA. As a control, proteinase K digestion was performed in each sample of untreated SI with 20 μg / ml at a protein to protein ratio of 1: 50 (Bolton, 1982) in the presence of either 0.15% Zwittergent, 0.2% or 2% of Sarkosyl. These reactions were terminated by the addition of 2 mM PMSF. The digested samples were centrifuged at 100,000 x g for 1 hour. The pellet containing all the insoluble proteins was resuspended in a minimum volume of PBS and 0.15% of Zwittergent. Subsequently, both the digested samples and the whole brain homogenate samples were sonicated in a bath sonicator in water for 20 minutes to denature the remaining protein after digestion. Each sample was then analyzed for the protein content of PrP by immunoblot. An aliquot of 100 μl of each sample was placed in a 1.5 ml Eppendorf tube with an equal volume of a sample charge regulator (1 X = 50 mM TrisCI, pH 6.8, 100 mM DTT, 2% of SDS, 0.1% bromophenol blue, and 10% glycerol). In addition, an aliquot of b-mercaptoethanol can be added to ensure denaturation of the proteins in the gel. The samples were run on a 10% polyacrylamide gel, molar ratio of bisacrylamide: acrylamide of 1: 29, for 15 V / cm for about 4 hours. The samples were boiled each for 10 minutes, and 15 μl of each sample was loaded onto the gel. For a more detailed description of separation of the protein by PAGE, see Schragger and von Jagow, nature 166: 368-379 (1987) and Laemmli, U.K. (1970) Nature 227, 680-385, both of which are incorporated herein in their entirety. The gel was removed from the PAGE apparatus and transferred to unloaded nylon (Amersham) by the use of an electrowinning apparatus. The gel and nylon were placed in sandwich between pieces of Whatman 3MM paper immersed in a transfer regulator containing Tris, glycine, SDS and methanol. The sandwich was placed between graphite plate electrodes, with the nylon on the anode side. A current of 0.65 mA / sq. Was applied. for 1.5-2 hours. After the transfer, the gel was stained with coomassie blue to ensure that the transfer was complete. The nylon filter was placed in a thermo-hermetic plastic bag and 0.1 ml of blocking solution per square cm of filter was added. The blocking solution contained 5% (w / v) of non-fat evaporated milk (Carnation); 0.01% antifoam A; and 0.02% sodium azide in PBS. After stirring 1 hour at room temperature, the monoclonal antibody 3F4 was added in a 1: 100 dilution, and the filter was incubated for 2-4 hours at 4 ° C with gentle agitation on a platform shaker. After incubation, the blocking solution and the antibody were removed and the filter was rinsed three times. The filter was incubated with a secondary anti-Ig antibody in blocking solution for 1-2 hours at room temperature. The secondary antibody was radiolabelled to allow immunoblot detection. For radiolabel probe I125, approximately 104 cpm of reagent per square centimeter of filter was added. After incubation, the filter was rinsed several times in PBS, with each rinse being approximately 10 minutes in length. The filter was placed in a cassette with a piece of Xomat X-ray film (Kodak) at -70 ° C. The results of the immunoblot were as follows: row 1 normal hamster S1 Result: strong band at approximately 33-35 Kd row 2 normal hamster S1 at 37 ° C Result: strong band at approximately 33-35 Kd row 3 normal hamster S1 digested with 1 00μg / ml dispase. 0.15% of Zwittergent Result: no band row 4 normal hamster S1 digested with 100μg / ml dispase. 0.2% of Sarkosyl Result: no band row 5 normal hamster S1 digested with 100ug / ml dispase, 2% Sarkosyl Result: no band row 6 normal hamster S1 digested with 20μg / ml Proteinase K, 0. 2% of Sarkosyl Result: no band row 7 normal hamster S1 digested with 20μg / ml Proteinase K, 2% Sarkosyl Result: no band row 8 hamster S1 infected with prion Result: strong band at approximately 33-35 Kd row 9 hamster S1 infected with prion Si at 37 ° C Result: strong band at approximately 33-35 Kd row 10 S1 hamster infected with digested prion with 100 μg / ml dispase, 0.15% Zwittergent Result: very strong band at approximately 33-35 Kd row 1 1 S1 hamster infected with digested prion with 100μg / ml dispase, 0.2% Sarkosyl Result: very strong band at approximately 33-35 Kd row 12 hamster S1 infected with prion digested with 100ug / ml dispase, 2% Sarkosyl Result: very strong band at approximately 33-35 Kd row 13 hamster S1 infected with prion digested with 20μg / ml protein kinase, 0.2% Sarkosyl Result: Very strong band at approximately 27-30 Kd row 14 S1 hamster infected with digested prion with 20uq / ml protein kinase. 2% of Sarkosyl Result: very strong band at approximately 27-30 Kd These results demonstrate that the normal hamster brain PrPc was not detected after digestion with Dispase or Proteinase K, while samples from the prion-infected brain showed a very strong signal of protease-resistant protein. Prion-infected samples, digested with proteinase K, showed an expected shift in the size of the molecular weight corresponding to the digestion of the n-terminus of the protein. Digestion with Dispase showed no shift in molecular weight: This finding showed that the Dispase is selective for the normal conformation of the protein. EXAMPLE 2 DETECTION OF PrPSc IN THE BRAIN OF MOUSE To determine the levels of PrPSc in affected mice, a mouse infected with prion and a normal mouse were each sacrificed and their brains were removed. A 10% (w / v) brain homogenate of normal and prion infected mice was prepared in PBS. After a low speed centrifugation at 500 X g for 15 minutes, the total protein in the supernatant (SI) was measured by the use of spectrophotometric assays and the concentration was adjusted to 2.5 mg / ml with PBS. The samples were digested with 500 U / ml Leucolysin for 45 minutes at 37 ° C in the presence of 2% Sarkosyl. Digestion was stopped by the addition of 50 mM EDTA. An aliquot of the proteins obtained in the SI both before and after the digestion of leucolysin were electrophoresed at 4 ° C on a polyacrylamide plate gel as described in the Laemmli reference, but in the absence of SDS and 2-mercaptoethanol . This allows the non-denatured protein to migrate through the polyacrylamide while preserving the native structure of the protein. Once immobilized in polyacrylamide, the gene proteins are then transferred to nitrocellulose for protein detection. The transfer may occur, as in Example 1, or a semi-dry transfer apparatus (Maniatis Reference) may be used. The digested and undigested sample of both mice, infected and normal, are detected in Western blot as described in Example 1, but using a monoclonal antibody that recognizes the native PrPSc form of the protein. Such antibodies are described in WO98 / 3741 1 published on August 27, 1998 and the U.S. Patent. 5,846,533 issued December 8, 1998, each of which is incorporated herein by reference in its entirety. Preferably, the antibody used is PrPSc-specific R1 antibody. The antibodies are added at a concentration of about 1: 100 to 1: 200, depending on the antibody used and the amount of protein predicted to be immobilized on the nitrocellulose. The nitrocellulose is placed in a thermo-hermetic plastic bag and 0.1 I of blocking solution is added per square cm of filter. The blocking solution contains 5% (w / v) evaporated non-fat milk (Carnation); 0.01% antifoam A; and 0.02% sodium azide in PBS, and Tween 20 was added to a final concentration of 0.02%. The Tween 20 is a mild detergent that also helps in reducing the above. After 1 hour of stirring at room temperature, the R1 antibody is added in a 1: 100 dilution and the filter is incubated for 2-4 hours at 4 ° C with gentle agitation on a platform shaker. After incubation, the blocking solution and the antibody are removed and the filter is rinsed three times. The filter is incubated with a secondary anti-Ig antibody in blocking solution for 1-2 hours at room temperature. The secondary antibody is conjugated with enzyme with horseradish peroxidase to allow detection by immunoblotting. The secondary antibody is added at a much higher diluted level than the primary antibody, at a dilution of from 1: 500 to 1: 2000. After incubation, the filter is rinsed several times in PBS, with each rinse being approximately 10 minutes in length. The filter is then placed in a cassette with a piece of Xomat X-ray film (Kodak) at -70 ° C. The resulting spotting will have a band corresponding to PrPSc in the mouse brain sample infected with prion, treated, both before and after digestion with Leucolysin. The sample from the normal mouse brain protein will show a low level band due to the binding of the PrPSc. The row with the normal brain sample subject to hydrolysis with Leucolysin, however, will not have a detectable band, since Leucolysine will have hydrolyzed the PrP protein in the sample. EXAMPLE 3 DETECTION OF PrPSc IN COW BRAIN A 10% (w / v) brain homogenate of normal and prion infected cows was resuspended in 1 L of 25 mM Tris-HCl, pH 8.0, 5 mM EDTA (regulator TO). This was centrifuged at 10,000 xg for 20 minutes and the supernatant containing soluble cytoplasmic proteins was discarded. The pellet was resuspended in 1 ml of regulator A, passed through a cellular switch twice (Microfluidics International, model MF1 10) and centrifuged at 30,000 xg for 1 hour, after which the supernatant was discarded and the pellet it was rinsed once in regulator A and centrifuged again at 30,000 xg for 1 hour. At this stage, the pellet could be stored at -20 ° C before hydrolysis. The digestion of β-lytic Metaloendopeptidase is carried out at a ratio of enzyme to protein of 1: 40. The protein is digested with 100 μg / ml β-lytic metalloendopeptidase (Sigma) for 75 minutes at 40 ° C in a pH 8.0 regulated solution containing 0.2% Sarkosyl. Digestion is stopped by the addition of 50 mM EDTA. After hydrolysis of the PrPSc conformation of the prion protein, the PrPSc can be denatured to allow the 3F4 antibody to recognize its epitope. Both samples, normal and prion infected, were treated with a 6M denaturing solution of guanidine HCl. The denaturation solution is prepared by diluting 10X of regulator (250 mM HEPES (pH 7.9); 30 mM MgCl 2; 40 mM KCl) with five volumes of distilled water. An appropriate amount of guanidine HCl is added and the solution is conducted to IX using distilled water. Finally, dithiothreitol is added to a final concentration of 1 mM. The hydrolyzed samples are subjected to treatment with the denaturation solution for 30 minutes at 4 ° C. The samples are then loaded onto 10% polyacrylamide gels as described in Example 1 and transferred to a nylon membrane. The nylon membrane is placed in a thermo-hermetic plastic bag and 0.1 ml of blocking solution per square cm of filter is added. The blocking solution contains 5% (w / v) nonfat evaporated milk (Carnation) and 0.02% sodium azide in PBS. After 1 hour of stirring at room temperature, the monoclonal antibody 3F4 is added at a dilution of 1: 200 and the filter is incubated for 2-4 hours at 4 ° C with gentle agitation on a platform shaker. After incubation, the blocking solution and the antibody are removed and the filter is rinsed three times. The filter is incubated with a secondary antibody of anti-Ig in blocking solution for 1-2 hours at room temperature, the filter is rinsed several times in PBS and placed in a cassette with a piece of X-ray film Xomat (Kodak ) at -70 ° C. The determination of the prion infection is based on a comparison of PrP recognition in the samples, normal and infected. The rows containing non-hydrolyzed S1 should contain relatively similar amounts of PrP protein recognized by the 3F4 antibody. The rows containing the samples treated with hydrolyzed guanidine HCl will allow the detection of PrPSc in the infected sample, since the only form left after hydrolysis is the PrPSc form. The ratio of the signal between the hydrolyzed and non-hydrolyzed sample of the infected cow will determine the percentage of PrP that is found in the conformation of PrPSc. The normal hydrolysed cow sample will later serve as a control that the conformation of PrPc was completed.

EXAMPLE 4 COMPARISON OF 3F4 AND DYEING R1 OF A SAMPLE To determine the levels of PrPSc in affected mice, a mouse infected with prion and a normal mouse were sacrificed and their brains were removed. A homogenate of 10% (w / v) of each of the brains was prepared by dispersing the brain tissue in PBS. The brain homogenate was then subjected to a low speed centrifugation of 500 x g for 15 minutes to separate the suspended proteins from unwanted cell debris. The total protein concentration of the supernatant (SI) was measured by the use of a BCA Protein Assay (Pierce) and the concentration of each brain homogenate was adjusted with PBS to 3.5 mg / ml. A portion of the homogenate was kept to serve as a control of the brain proteins in total. Each sample was then analyzed for the protein content of PrPSc and PrPc by immunostaining. Two aliquots of 100 μl of each sample were processed and electrophoresed at 4 ° C on an 8% polyacrylamide plate gel, as described in Example 2, ie, under non-denaturing conditions. The gel is loaded with two rows of sample of the affected mouse and two rows of sample of the control mouse and preferably one row of each is passed on one side of the gel, one row of each on the other side of the gel, without containing the cavities show some that separates the two sides. The gel is passed and transferred to the nylon as in Example 1. After transfer, the nylon is cut to separate the nylon into two separable spots, each row containing an affected sample and one row of control sample. Each nylon filter is placed in a thermo-hermetic plastic bag and 0.1 ml of blocking solution per square cm of filter is added. The blocking solution contains 5% (w / v) nonfat evaporated milk (Carnation) and 0.02% sodium azide in PBS. After 1 hour of stirring at room temperature, the 3F4 antibody is added to a spot at a dilution of 1: 200 and R1 antibody is added to the other spot at a dilution of 1: 200 and the filter is incubated 2-4. hours at 4 ° C with gentle agitation on a platform shaker. After incubation, the blocking solution and the antibody are removed and each filter is rinsed three times. The secondary antibody is appropriately labeled to allow detection of immunoblotting. The secondary antibody is added at a much more dilute level than the primary antibody, at a dilution of 1: 500 to 1: 2000. After incubation, the filter is rinsed several times in PBS, with each rinse being approximately 10 minutes in length. The filter is then placed in a cassette with a piece of Xomat X-ray film (Kodak) at -70 ° C. The level of PrPSc in the affected sample can be determined by comparing the relative levels of the signal of each antibody in the affected mouse versus the control mouse. This process can be a physical comparison to determine the relative differences in the signal level between R1 and the 3F4 antibody, or it can be a quantitative comparison. The comparison techniques, both physical and quantitative, will be known by those experts in the field. The quantitative comparison can be determined through the use of spotting exploration techniques in which the levels are determined through the use of specially designed computer programs to determine comparative levels. One such method uses a modified Excel spreadsheet program. Such programs allow adjustments between samples based on variables such as background, hybridization time, etc. The staining levels of 3F4 should be consistent between the normal control samples of both stained with antibody R1 and 3F4. The level of difference between the R1 dyeing and the 3F4 staining in the affected sample should allow the quantification of PrPSc in the sample by subtracting the level of the PrPc signal using the 3F4 antibody from the PrPSc level and the PrPc signal from the R1 antibody . Such quantification can be adjusted based on the sensitivity of the antibody, concentration, etc. EXAMPLE 5 DETECTION OF ßA4 IN HUMAN AND MOUSE BRAINS A number of mouse models of Alzheimer's disease exhibit many of the contrast protein changes associated with human disease. Two examples are: 1) mice with a modified human APP under the control of the PDGF promoter (the "Athena-Lilly mouse"), the production and phenotype of these mice are described in US Patent No. 5, 612, 486 and 2) mice with a mutant isoform of human APP under the control of the prion genetic promoter (the "Hsiao mouse") see Hsiao et al., Science 274: 99-102 (1996). These mouse models for Alzheimer's disease show amyloid deposits and are capable of producing the three main isoforms of APP and the levels of APP and Aß40 / Aβ42 that increase progressively during the lifetime of the mouse. A brain homogenate of 10% (w / v) of normal mice, affected Hsiao mice and affected Athena mice was prepared in TBS (25 mM Tris). After a low speed centrifugation at 500 xg for 15 minutes, the total protein in the supernatant (SI) was measured by using a BCA Protein Assay (Pierce) and the concentration was adjusted to 2.5 mg / ml with PBS . A digestion of Dispase is done at a ratio of enzyme to protein of 1: 25. The protein is digested with 100 μg / ml of Dispase (Worthington) for 60 minutes at 37 ° C in the presence of 0.15% of Zwittergent 3-12 (Calbiochem). Digestion is stopped by the addition of 50 mM EDTA. PAGE and transfer of the proteins is carried out as in Example 1. The nylon filter is placed in a thermo-hermetic plastic bag and 0.1 ml of blocking solution is added per square cm of filter. The blocking solution contains 5% (w / v) of non-greasy evaporated milk (Carnation); 0.01% antifoam A; and 0.02% sodium azide in PBS. After 1 hour of stirring at room temperature, a monoclonal antibody which recognizes the β-amyloid protein, such as RDI-BAMILOIDE (Research Diagnostics) is added at a dilution of 1: 200 and the filter is incubated for 2-4 hours at 4 ° C with gentle agitation on a platform shaker. After incubation, the blocking solution and the antibody are removed and the filter is rinsed three times. The filter is incubated with a mouse anti-Ig secondary antibody in blocking solution for 1-2 hours at room temperature. The secondary antibody is radiolabelled to allow detection of immunoblotting. For the radiolabel probe I125, approximately 104 cm of reagent per square centimeter of filter was added. After incubation, the filter was rinsed several times in PBS, with each rinse being approximately 10 minutes in length. The filter was placed in a cassette with a piece of Xomat X-ray film (Kodak) at -70 ° C. The resulting spotting will have a band corresponding to ßA4 in the sample of the mouse brain Athena Lilly or Hsiao treated, both before and after digestion with Dispase. The normal mouse brain protein sample may show a low level band due to the background with the APP protein. The row with the normal brain sample subject to hydrolysis with Dispase should not have a detectable band since the Dispase will have hydrolysed the normal protein in the sample. This protocol can also be used with biopsy material or human brain, to identify and diagnose individuals suspected of having Alzheimer's disease. EXAMPLE 6 TTR DETECTION IN BIOPSY MATERIAL A biopsy sample is taken from the liver of an individual who is thought to suffer from familial amyloid polyneuropathy (FAP). The homogenates (10% (w / v)) of the liver sample and a normal liver control sample are prepared in TBS (25 mM Tris). After a low speed centrifugation at 500 xg for 15 minutes, the total protein in the supernatant (SI) is measured by using a BCA Protein Assay (Pierce) and the concentration is adjusted to 3.5 mg / ml with PBS . A digestion of Neprilysin is carried out at a ratio of enzyme to protein of 1: 35. The protein is digested with 100 μg / ml of Dispase for 60 minutes at 37 ° C in the presence of 0.2% Sarkosyl. Digestion is stopped by the addition of 50 mM EDTA. An aliquot of the proteins obtained in SI both before and after the digestion of neprilysin is electrophoresed at 4 ° C on an 8% polyacrylamide platform gel, as described in Example 2, that is, under non-denaturing conditions. The gel is passed and the proteins are transferred to nylon as in Example 1. The nylon filter is placed in a thermo-hermetic plastic bag and 0.1 ml of blocking solution is added per square cm of filter. The blocking solution contains 5% (w / v) nonfat evaporated milk (Carnation) and 0.02% sodium azide in PBS. After 1 hour of stirring at room temperature, a monoclonal antibody recognizing the amyloid conformation of TTR is added at a dilution of 1: 100 to 1: 500 and the filter is incubated for 2-4 hours at 4 ° C with gentle shaking on a platform agitator. After incubation, the blocking solution and the antibody are removed and the filter is rinsed three times. The secondary antibody is conjugated with enzyme with horseradish peroxidase to allow the detection of immunoblotting. The secondary antibody is added at a much more dilute level than the primary antibody, at a dilution of from 1: 500 to 1: 2000. After incubation, the filter is rinsed several times in PBS, with each rinse being approximately 10 minutes in length. The filter is then placed in a cassette with a piece of Xomat X-ray film (Kodak) at -70 ° C. The resulting staining will have a band corresponding to the TTR amyloid conformation in an affected biopsy sample., but not in a biopsy sample in which amyloids do not occur. In addition, the concentration of the amyloid conformation of TTR can be an indicator of prognosis of the severity of the disease, and biopsies using multiple samples can also be used to determine the progress of the disease. The present invention is shown and described herein in what is considered to be the most practical and preferred modalities. However, it is recognized that departures from them may be made within the scope of the invention and those skilled in the art will come up with obvious modifications after reading the disclosure.

Claims (19)

1. CLAIMS A test method, characterized in that it comprises the steps of: treating a sample suspected of containing a protein that assumes a first conformation and a second conformation related to the disease, with a compound that hydrolyzes the protein in the first conformation but not in the second conformation related to the disease in order to provide a treated sample; contact the sample in treatment with a link partner that joins the conformation related to the disease; and determine the presence of the conformation related to the disease based on the linkage partner's linkage.
2. 2. The test method according to claim 1, characterized in that the protein is a PrP protein, the first conformation is PrPc and the second conformation related to the disease is PrPSc and the compound that hydrolyzes the PrPc changes the conformation of the PrPSc in order to that an epitope not previously exposed is exposed after treatment.
3. 3. The test method according to claim 2, characterized in that the compound that hydrolyzes PrPc but not PrPSc is selected from the group consisting of metalloendopeptidase and Dispase.
4. The test method according to claim 1, characterized in that it further comprises: separating the protein from the second disease-related conformation of the treated sample; contact the separated portion with the linking partner.
5. The test method according to claim 4, characterized in that the separation is carried out by centrifugation and the binding partner is an antibody and wherein the protein is selected from the group consisting of a protein of PrP, a protein of TTR and an amyloid protein.
6. The test method according to claim 1, characterized in that it further comprises: pretreating the sample before treating the sample with the compound that hydrolyzes the protein in the first conformation, wherein the pretreatment reduces the protein concentration in the sample different from the conformation related to the disease of the protein.
7. The test method according to claim 6, characterized in that the pretreatment comprises the removal of the protein in the sample by contacting the sample with antibodies bound to a support, whose antibodies bind to the protein other than the protein in the sample. second conformation related to the disease.
8. 8. The test method according to claim 6, characterized in that the pretreatment comprises the hydrolyzation of proteins in the sample different from the proteins in the second conformation related to the disease.
9. 9. The test method according to claim 1, characterized in that the binding partner is an antibody of R1. 1 0.
10. An assay method, characterized in that it comprises the steps of: treating a sample suspected of containing a protein that assumes a first conformation and a second conformation related to the disease, with a compound that hydrolyzes the protein in the first conformation but not in the second conformation related to the disease to provide a treated sample; denaturing the protein in the second conformation related to the disease in order to provide a denatured, treated sample; contact the denatured sample, treated, with a binding partner that joins the second conformation related to the disease, denatured, of the protein; and detect the second conformation related to the disease of the protein based on the linkage to the partner. eleven .
11. The test method according to claim 10, characterized in that the protein is a protein selected from the group consisting of PrP protein, a TTR protein and an amyloid protein.
12. The test method according to claim 10, characterized in that the protein is a PrP protein, the first conformation of the protein is PrPc and the second conformation related to the disease of the protein is PrPSc.
13. The test method according to claim 10, characterized in that the protein in the first conformation is PrPc and the compound that hydrolyzes PrPc is selected from the group consisting of Dispase and metalloendopeptidase
14. 14. The test method according to claim 10, characterized in that the binding partner is attached to a label 5 detectable.
15. 15. The test method according to claim 14, characterized in that the detection is carried out by the use of improved fluorescence by dissociation, resolved by time.
16. 16. The test method according to claim 14, characterized in that the detection is carried out by the use of a double-wavelength laser-activated fluorometer.
17. 17. A test method, characterized in that it comprises the steps of: providing a sample suspected of containing a protein that assumes a first conformation and a second conformation related to the disease; contact the sample with a first link partner, whose first link partner joins the first conformation but not the second conformation related to the disease of the protein, in 20 where the first link partner is added in sufficient quantities to join to substantially all the epitopes available in the sample; contacting the sample with a second binding partner that binds both to the first conformation of the protein and to the second conformation related to the disease of the protein; and detecting the presence of the second conformation related to the disease of the protein based on the link to the second partner of linkage.
18. 18. The assay according to claim 17, characterized in that the first binding partner is a 3F4 antibody.
19. 19. The test method according to claim 17, characterized in that the second binding partner is an R1 antibody.