MXPA99007739A - Assay for disease related conformation of a protein - Google Patents

Abstract

An assay method is disclosed which makes it possible to determine the presence of a diseased related conformation of a protein (e.g., PrPSc or the&bgr;-sheet form of&bgr;A4) in a sample. A sample is divided into two portions and the first portion is cross-linked to a first solid support and then contacted with a labeled antibody which binds to a non-disease form of the protein with a higher degree of affinity (e.g., 4 to 30 fold higher) than to the disease form of the protein to change conformation to a form with a higher binding affinity for the labeled antibody. The treated second portion is then bound to a second solid support and contacted with labeled antibody. The level of labeled antibody binding to a protein in the first and second portions is determined and the amounts measured in each are compared. The difference between the two measurements is an indicationof whether the disease related conformation of the protein was present in the sample. The method can also determine the concentration of the disease related conformation and the particular strain present.

Description

TEST TO DETERMINE CONFORMATION RELATED TO PROTEIN DISEASES FIELD OF THE INVENTION This invention relates in general to immunoassays. More particularly, the invention relates to an assay, which allows the detection of a conformational form of a disease of a protein (such as PrPSc), which may have low binding affinity for antibody and which additionally allows the identification of the particular strain responsible for the disease.

BACKGROUND OF THE INVENTION Prions are infectious pathogens that invariably cause 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 nucleic acid is not needed to allow the infectivity of a prion protein to proceed. A major step in the study of prions and the diseases they cause was the discovery and purification of a protein called prion protein [Bolton, McKinley et al. (1982) Science 218: 1309-1311; Prusiner, Bolton et al. (1982) Biochemistry 21: 6942- 6950; McKinley, Bolton et al. (1983) Cell 35: 57-621. Since then, the complete prion protein coding genes in transgenic animals have been cloned, sequenced and expressed. PrPc is encoded by a single copy host gene [Basler, Oesch et al. (1986) Cell 46: 417-428] and when PrPc is expressed, it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases are the result of the transformation of the normal form of the prion protein (PrPc) into the abnormal form (PrPSc). There is no difference that can be detected in the amino acid sequence of the two forms. However, PrPS (r), when compared to PrPc, has a higher content of / S-lamellate and lower a-helix [Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90: 10962-10966 Safar, Roller et al. (1993) J Biol Chem 268: 20276-202841 The presence of the abnormal PrPSc form in the brains of humans or infected animals is the only disease-specific diagnostic marker of prion diseases. PrPSc plays a key role both in the transmission and in the 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 the scraping of sheep and goats and cattle bovine spongiform encephalopathy (BSE) [ilesmith and Wells (1991) Curr Top Microbiol Immunol 172: 21-38]. Four prion diseases have been identified in humans: (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Streussler-Sheinker disease (GSS) in English), and (4) fatal family insomnia (FFI, for its acronym in English) [Gajdusek (1977) Science 197: 943-960; Medori, Tritschler et al. (1992) N Encrl J Med 326: 444-449]. Initially, the presentation of human inherited prion diseases posed an intricate question 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, 2a. edition: 165-186]. The strains are differentiated by the incubation time, by the topology of accumulation of the PrPSc protein, and in some cases also by the distribution and the characteristics of the cerebral pathology.

[DeArmond and Prusiner (1997) Greenfield's Neuropathology, 6a. edition: 235-280]. Because the PrPSc is the principal, and most likely, the only prion component, the existence of the prion strains has raised an issue intricate as to how biological information can be encoded in a molecule that is not one that comprises nucleic acids. It has been found that partial proteolytic treatment of brain homogenates containing some prion isolates generates peptides with slightly different electrophoretic variabilities [Bessen and Marsh (1992) J Virol 66: 2096-2101; Bessen and Marsh (1992) J Gen Virol 73: 329-334; Telling, Parchi et al _ (1996) Science 274: 2079-2082]. These findings suggested different sites of proteolytic dissociation, due to the different conformation of the PrPSc molecules in the different prion strains. Alternatively, one could explain the differences that were observed by the formation of different complexes with other molecules, which form different dissociation sites in the PrPSc in the different strains [Marsh and Bessen (1994) Phil Trans R Soc Lond B 343: 413-414]. Some investigators 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 the patterns of both glycosylation and peptide mapping in the diagnosis of multiple prion strains is still under debate [Collings, Hill et al. (1997) Nature 386: 564; Somerville, Chong et al. (1997) Nature 386: 564].

In Serban et al., Neurology, volume 40, no. 1, Ja 1990, a system for detecting PrPSc is provided by intensification of the immunoreactivity after denaturation. Sufficient sensitivity and specific direct assay for PrPSc in biological samples could potentially eliminate the need for inoculations of the animal completely. Unfortunately, this does not seem possible with current PrPSc assays - it is estimated that the current sensitivity limit of PrPSc detection, which is based on proteinase K and Western blotting, is in the range of 1 μg / milliliter , which corresponds to 104-105 infectious units of prions. Additionally, the specificity of the traditional proteinase K-based assays for PrPSc is questioned in light of recent findings of the relative only or non-K proteinase resistance of the undoubtedly infectious prion preparations [Hsiao, Groth and collaborators (1994) Proc Natl Acad Sci USA 91: 9126-9130] Telling et al. (1996) Genes & Dev. Human transthyretin (TTR) is a normal plasma protein composed of four structured units, predominantly of identical β-lamellae, and serves as a hormone thyroxine transporter. The same abnormal assembly of the TTR in amyloid fibrils, causes two forms of human diseases namely, Senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) [Kelly (1996) Curr Opin Strut Biol 6 (1) -.11-71. The cause of amyloid formation in FAP are point mutations in the TTR gene; The cause of the SSA is unknown. The clinical diagnosis is established histologically by detecting amyloid deposits in situ in bioptic material. Until now, little is known about the mechanism of the conversion of TTR to amyloid in vivo. However, different laboratories have demonstrated that amyloid in vitro conversion can be stimulated by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993) Biochemistrv 32 (45): 12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun 197 (2): 415-21]. The mechanism of conformational transition includes the monomeric conformational intermediate, which is polymerized in structured 3-lamellar amyloid fibrils [Lai, Colon et al. (1996) Biochemistry 35 (20): 6470-82]. The process can be mitigated by fixation with stabilizing molecules, such as thyroxine or triiodophenol [Miroy, Lai et al. (1996) Proc Natl Acad Sci USA 93 (26) .15051-6]. In view of the previous points, there is clearly a need for a specific trial, of high affluence, and cost effective, to test sample materials for the presence of the pathogenic protein, including transthyretin and prion protein. The invention presented offers an assay that can not only detect pathogenic proteins, but determine the specific strain.

COMPENDIUM OF THE INVENTION There are two different procedures for detecting low levels of a disease conformation of a protein. The simplest method requires that a predetermined standard has been calculated, and comprises providing a sample suspected of containing a protein that assumes two conformations (a first conformation, and a second conformation related to diseases), treating the sample to transform any protein into the second conformation, into a different conformation having a higher antibody binding affinity, by contacting the treated sample with an antibody, which binds to the protein in its first conformation and / or conformation different with a higher affinity than the second conformation, determining the level of binding of the antibody to the protein, and comparing the level of binding to the standard that was previously determined, determining by the same the probability that the sample contains the protein in the second conformation related to diseases, based on the comparison. The methodology described herein allows the detection of disease conformation at a level of 1 'x 1.O3 particles / milliliter or less. The assay of the invention can also be conducted without the use of a previously determined standard. This test method comprises (a) providing a sample suspected of containing the protein (having a first conformation and a second conformation related to diseases), (b) dividing the sample into a first and second portions, (c) ) contacting the first portion with an antibody that binds to the first conformation with higher affinity than the second conformation, (d) treating the second portion to cause any protein in the second conformation, to adopt a different conformation having a higher affinity for the antibody, (e) contacting the second portion with the antibody, (f) determining the relative levels of antibody binding to the first and second portions, and (g) determining the presence or absence of the antibody. protein in the second conformation, based on the comparison. The assay of the invention is useful when testing samples containing proteins, which are present in at least two conformations (eg, a native conformation without disease and a disease conformation) and are present at levels of 1 x 103 particles / milliliter or less. The present invention utilizes antibodies that do not bind or have a relatively low degree of affinity for tightly configured disease conformation of the protein. A useful antibody is the monoclonal antibody 263K 3F4 which produces the hybridoma ATCC HB9222 cell line, which was deposited on October 8, 1986 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 and which is described in FIG. U.S. Patent No. 4,806,627, issued February 21, 1989 - which is incorporated by reference to describe antibodies that selectively fix PrPc. A specific example of the assay can be made by providing a sample that is divided into a first portion and a second portion. The first portion is fixed to a first solid support and then contacted with a labeled antibody, which binds to the disease free form of the protein with a higher degree of affinity (for example, from 4 to 30 times of higher affinity), than the antibody that binds to the disease form. The second portion of the sample is traced in a manner that causes the tightly fixed form of the disease of the protein present in the sample (if any) to assume a more relaxed conformation that has a higher binding affinity to the labeled antibody. After the treatment, it is also fixed the second portion to the surface of a solid support. After this, the second portion is contacted with the same type of labeled antibodies that were used in the first portion. Then, based on the amount of antibody binding protein in the support, the level of protein in the first portion is determined. The level of protein in the second portion that was treated is determined in the same way. The difference between the two is determined, and if it is found that the amount of antibody binding to the proteins in the second portion is significantly greater than that of the first portion, it is possible to deduce that the original sample contained proteins in the conformation related to diseases fixed tightly. In addition, by using the formulas and a particularly sensitive assay system that is provided herein, it is possible to determine the amount of conformation related to diseases of the protein present in the original sample per unit volume. Still further, by determining the rate of binding of the antibody to the denatured protein to the binding of the antibody to the native protein, it is possible to determine the particular strain of a protein the disease present. To demonstrate the basic concept behind the present invention, it is necessary to include a start sample, which is divided into at least two portions. It gets in contact the first portion with the labeled antibodies, without treating the proteins and treat the second portion with the labeled antibodies after the proteins have been treated in a way that causes any of the proteins in the conformation of disease to assume a conformation with a higher degree of binding affinity for the antibodies. The readings are compared (that is, one subtracted from the other) and the presence of proteins in the conformation related to diseases is deduced, based on the difference between the two readings. However, it is possible to use the basic concept behind the present invention, without obtaining two readings for each test. This can be done by establishing a standard that is based on the performance of the trial on a statistically significant number of closely related samples. After the standard has been established, one will know the level of antibody binding that should be observed when the sample does not contain any protein in the disease-related conformation. Using the standard, one treats a sample to be tested in a manner to convert any proteins in the conformation related to diseases in a different conformation, which has a much higher degree of binding affinity for the labeled antibodies. Then the measure that was obtained with the standard is compared. If the difference between the standard and the measure that was obtained is outside a given range, it can be deduced that the original sample included proteins in the conformation related to diseases. A third embodiment of the invention can use any of the embodiments described above with the formulas provided herein, with the aim of calculating (quantitatively) the number of proteins in the conformation related to diseases present within the original sample. In the fourth embodiment, the particular strain of the infectious protein present in the sample is determined. The strain is determined by matching the "protein index" of the sample that was tested, with the known protein index of the strain of the particle of a given protein. The "protein index" is calculated by determining the proportion of the amount of the antibody that binds the denatured form of the protein to the amount of the antibody that binds to the native protein. In accordance with any of the systems, it is preferable to pretreat the sample being treated, in a manner that decreases the concentration of the disease-free conformation in relation to the disease conformation of the protein. For example, the initial sample can be chemically treated with a compound that preferably degrades the relaxed form, without disease of the protein and / or is exposed to the antibodies to which it is preferably fixed and remove by the same the disease-free conformation of the protein. It might be possible to further increase the sensitivity of the different aspects of the invention, by concentrating the disease conformation of a protein, by adding a compound that is selectively bound to the disease conformation, to form a complex and subject the sample to centrifugation to precipitate the complex, which is then tested, in accordance with the methods described herein. In our lawyer registration number 6510/098001 of the pending application, issued on the same date as the present application entitled "Process for Concentrating Protein with Disease-Related Conformation," the specific ones are described in detail with respect to these methods of concentration . The different embodiments of the assay of the invention described above are all "direct" types of immunoassays - meaning that the sample is tested directly with the labeled antibody, either with or without treatment to change the conformation of any conformation proteins related to diseases present in the sample. An "indirect" test can also be used. For example, it may be desirable to increase the number of proteins related to diseases in the sample (if any), by using a transgenic mouse and intensifying any signal obtained by the same. To perform these embodiments of the invention, the sample is first used to inoculate a transgenic mouse, from which its genome has been modified so that it will develop disease symptoms when it is inoculated with the proteins in the conformation diseases. After the mice are inoculated, a sufficient period of time is allowed (e.g., 30 days), after which the transgenic animal is sacrificed and a sample, such as a mouse homogenate brain tissue, is used in the direct trial described above. The present invention enhances the ability of transgenic mice to detect prions, by shortening the period of time that must elapse before the determination can be made, as to whether the original sample contained proteins in the disease-related conformation. It would also be possible to apply the epitope tagged PrP, as described in pending U.S. Patent Application Serial No. 08 / 660,626, which was filed on June 6, 1996 (incorporated by reference) for affinity purification. PrPSc from the brain of a mouse. Tg and apply by the same the assay of the present invention. Without the present invention, the mouse and one are inoculated you must wait until the mouse that is inoculated actually shows the symptoms of the disease. Depending on the mouse, this can take several months or even years. 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 binding affinity, so that the two conformations can be distinguished by the use of antibodies or binding partners, which may have a higher degree of affinity for the first conformation than for the second conformation. In its simplest conceptual form, the invention works best when a known labeled antibody binds to a disease-free form of a protein with a high degree of affinity, and does not bind (or bind with an extremely low degree of affinity) ) to the same protein when it is present in its conformation related to diseases. However, in reality, a given protein can have more than two conformations. The protein can have more than one conformation without disease and more than one conformation related to diseases, (Telling and collaborators, Science (1996)). The invention is still useful when there are multiple conformations of disease free and disease forms of the protein - provided that (1) at least one conformation without disease differs from at least one disease conformation in terms of its binding affinity; and (2) it is possible to treat the conformation related to diseases of the protein in order to substantially enhance its binding affinity. As indicated above, the assay of the invention can be used to test any type of sample for any type of protein, provided that the protein includes a conformation without disease and diseases. However, the invention was developed in a particular manner to test samples to detect the presence of (1) PrP proteins and to determine if the sample included a PrP protein in its disease conformation, ie, if it included the PrPSc, (2) forms 3A4 insolubles associated with Alzheimer's disease and (3) transthyretin. In accordance with the above, most of the following description is directed to the use of the immunoassay of the present invention, to detect the presence of PrPSc (or to a lesser degree, / 3A4 or transthyretin (TTR)) in a sample - it being understood that the same general concepts are applicable to detect conformations related to diseases of a wide range of different types of proteins. In addition, the description is directed in a particular to the description of how to determine the particular strain of infectious prions (PrPSc) in a sample - it being understood that the same general concepts are applicable to determine the particular strain of other tight proteins associated with different diseases. The present detection method of PrPSc was developed by labeling purified IgG selected with Europium. The antibodies that are used have a high binding affinity for PrPc (conformation without disease), which comprises an a-helical configuration. The antibodies have a low binding affinity for PrPSc (disease conformation), which comprises a β-lamella configuration. IgG can be obtained from common monoclonal, polyclonal, or recombinant antibodies, typically recognizing the 90-145 or 222-231 sequence of the PrPc and the prion protein cleaved conformationally. Different conformations of recombinant prion protein were chemically crosslinked to polystyrene plates through an activation step of glutaraldehyde. The relative affinities of the Eu-labeled IgG with the α-helical, the lamellar, and the random serpentine conformation of the recombinant prion protein of the Syrian hamster corresponding to the sequence 90-231 were determined by fluorescence resolved by time, of improved dissociation in a polystyrene plate format of 96 cavities A determination of the relative affinities of the different antibodies labeled for the proteins can be made by different methods. However, the conformation related to diseases of a protein is frequently present in a very low concentration in relation to that of the conformation without disease. In accordance with the foregoing, very sensitive methods are often required to detect any increase caused by the treatment of the disease conformation of the protein. A particularly sensitive method is the time resolved, improved dissociation fluorescence. By carefully calibrating this method, it is possible to detect the increase of the signal in the reactivity of the antibody in the transition from the conformational state of / 3-lamella to the denatured state. This signal is relatively large compared to that which was obtained from the transformation from its native a-helical to the relaxed state treated, or denatured. In this way, the original conformational state can be assigned by differential testing in the native and treated states. When a sample that does not contain the ß-lamella protein is treated, some increase in immunoreactivity is obtained. This amount of increase must be adjusted and after doing this, reference is made to the resulting concentration or amount as the "adjusted amount". The amount of antibody specific binding over which was obtained for the helical conformation of the PrPc (beyond the adjusted amount), is a measure of the presence of the 6-lamellar conformation, which is essential for pathogenicity and infectivity of the PrPSc. A first objective of the invention is to provide an immunoassay, which is applicable to assay samples containing proteins, samples of which are suspected to contain a protein that occurs within a conformation without native disease and a conformation related to diseases (e.g. , PrP protein, protein / 3A4 and transthyretin). An advantage of the present invention is that the immunoassay can quickly and accurately determine the presence of proteins in the disease-related conformation (e.g., PrPSc, 0A4 and transthyretin), although the antibody that was used does not bind or have a very low degree of binding affinity for the protein in the disease-related conformation, and the disease-related conformation is present in a lower concentration than the disease-free conformation. Another objective of the invention is to provide an assay that makes it possible not only to determine (1) whether the pathogenic particle is present or not in a sample, but (2) determine the concentration of the particles in a sample, and (3) determine the particular strain of the particle present. A feature of the invention is that the signal that was obtained can be enhanced 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 time resolved, enhanced dissociation fluorescence is used to intensify sensitivity. Another advantage is that the assay can detect the disease conformation levels of a protein at a concentration of 1 x 103 particles / milliliter or less. A specific objective is to provide a diagnostic assay to determine the presence of the infectious prion protein in variable sample materials, which were obtained or derived from tissues and / or body fluids of human, primate, monkey, pig, bovine , sheep, deer, elk, cat, dog, mouse and chicken. Another specific objective is to provide a diagnostic assay for determining the presence of the 3A4 protein in variable sample materials that were obtained or derived from tissues and / or body fluids of human, primate, monkey, pig, bovine, sheep, Deer, elk, cat, dog, mouse and chicken. Another objective is to provide a rapid test for detect the native infectious prion protein in the brains of transgenic and non-transgenic animals that were injected with sample material potentially containing prions. Another objective is to provide a method for evaluating decontamination procedures by testing the level of denaturation of the pathogenic proteins (e.g., prions or ßA4 of ^ -laminilla), after these treatments. Another objective is to provide a rapid method for screening different compounds to assess their potential to treat diseases associated with the disease conformations of different proteins, such as by screening the compounds for their stabilizing effect on the different conformations of the protein ( example, PrPc or the a-helical conformation of 3A4) or its destabilizing impact on the pathogenic conformation (for example, PrPSc or the β-lamella conformation of the A4) of a protein. Another objective is to provide a rapid method for screening different pharmaceutical compounds with potential for the treatment of prion disease, such as by screening the compounds for their stabilizing effect on the α-helical conformation of a normal isoform of the PrPc protein, or its destabilizing impact on the ß-lamella conformation of the pathogenic isoform of the PrPSc protein. Another advantage is that you can perform the process without an antibody directly capable of recognizing an infectious conformation of a protein, and without using a step of proteinase K to remove the signal from the normal isoforms (without disease) of the protein, such as PrPc. "Another advantage is that in the process that was invented, there is no need for the antibody to be able to directly recognize the pathogenic conformation of / 3A4 or transthyretin.An important feature of the assay is the rapid design, cost effective and of high affluence that can be designed with the ability to track 96 samples per day per 96-well plate Another aspect of the invention is the diagnostic method to quantitatively detect TTR in the abnormal conformation, amyloid in the material of shows that it was obtained from human and animal tissues, body fluids, and pharmaceuticals.The process that was invented provides a direct, sensitive method to distinguish and quantify the normal and amyloid conformations of the TTR in a mixture present in the materials of The quantification is based on the measurement of the difference in the affinities of the monoclonal and polyclonal bodies with TTR in the conformation. normal or amyloid, compared to random serpentine conformation. The present invention describes three evaluation methods and the mathematical formula that was used for this quantification. An important objective is to provide the specific diagnostic assay for the pathogenic TTR in variable sample materials that were obtained or derived from tissues of human, primate, monkey, pig, bovine, sheep, deer, elk, cat, dog, mouse and chicken. Another objective is to provide a rapid assay to detect the amyloid form of TTR in transgenic animals. Another objective is to provide a rapid method for screening different pharmaceutical compounds with potential for 'the treatment of senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). These compounds are screened for their stabilizing effect on the normal conformations of the TTR or their destabilizing impact on the amyloid conformation of the TTR. Still another objective is to provide a rapid method to track the impact of different spontaneous and designed mutations in the TTR gene on the conformation, stability and amyloid formation of these TTR gene products in transgenic animals harboring natural APP genes. or artificial. The specific advantage is that the assay that was invented can detect a pathogenic form of the TTR in a mixture with denatured nonpathogenic forms thereof or in a mixture with a soluble form of the TTR - for example, detect less than 1 x 103 particles per milliliter. These and other objects, advantages, and features of the invented process will be apparent to those skilled in the art, after reading the details of the test method, development and testing of the antibody, and the transgenic mouse, as described in more detail below. with reference to the figures that are attached.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a spectrography of SHaPrP90-231 conformation determined by circular dichromatism (CD) spectrography, which shows the two main bands with the minimum in 208 and 222 nm which "indicates an a-helical conformation, the single negative band with the minimum at 217 nm which is characteristic of the predominantly / S-lamellate conformation; Figure 2 is a graph showing the results of the SHaPrP90-231 competitive assay in the a-helical and denatured conformations, in the presence of 5% PrP0'0 mouse brain homogenate, where the difference in tilt and transverse points obtained with the 3F4 IgG labeled with Europium indicate that each of the conformations has both an affinity and a number of different binding sites and where additionally, the points and the data bars represent the mean ± SEM that was obtained from the four independent measurements; Figure 3 is a graph showing the calibration of a direct assay with SHaPrP90-231 in the α-helical conformation, in the presence of 5% PrP0'0 mouse brain homogenate, where the dots and bars Data represent the mean ± SEM that was obtained from the four independent measures; Figure 4 is a graph showing the calibration of a direct assay with SHaPrP90-231 in the ß-lamella conformation, in the presence of mouse brain homogenate of prp? / Or a1_ 5 p? R C in o, in where the data points and bars represent the mean ± SEM that was obtained from the four independent measurements; Figure 5 is a graph showing the input-output validation of a direct assay for the both a-helical and / 3-lamella forms of SHaPrP90-231 in the presence of mouse brain homogenate from PrP0'0 to 5 percent, where the amount of the protein on the x axis was determined by the amino acid analysis and the amount of the protein was calculated on the axis and from the test; Figure 6 is a graph showing the ratio between the signals of the SHaPrP90-231 treated (denatured) and native in the a-helical and / 3-lamella conformations, which were developed with Eu-labeled 3F4 IgG, where the data points and bars represent the mean ± SEM that was obtained from the four independent measures; Figure 7 is a graph showing the results of a direct assay for the PrPc protein in normal hamster brain homogenate, where the dots and data bars represent the mean ± SEM that was obtained from the four independent measurements; Figure 8 is a graph showing the results of a direct assay for prp + Sc in scraped-infected hamster brain homogenate, where the dots and data bars represent the mean ± -SEM that was obtained from the four independent measures; Figure 9 is a graph showing the total amount of PrP proteins in normal hamster brains and the amount of 3-lamellar PrPS in the two brains that was calculated from the model, - where the data points and bars represent the mean ± SEM that was obtained from the four independent measures; Figure 10 is a graph showing the total amount of PrP proteins in scrapie-infected hamster brains and the amount of β-lamellar PrPSc in the two brains that was calculated from the model, where the dots and bars data represent the mean ± SEM that was obtained from the four independent measures; Figure 11 is a graph showing the correlation of the infectivity and the amount of the ß-lamella form of SHaPrPSc, as calculated from the direct assay and the formula, where it was made sonic in the presence of mouse brain homogenate from PrP0'0 to 5 percent and diluted as described, and where the data points and bars represent the mean ± SEM that was obtained from the four independent measures; Figure 12 is a graph of 0A4 (1-40) in both the a-helical and / 3-lamella conformations that was produced by circular dichromatism (CD) spectroscopy, showing the major links at 208 and 222 nm for the helical conformation and a single negative band a.217 nm for the predominantly -lamellar conformation (at a pH of 7.4); Figure 13 is a graph of a direct test of A43 (1-40); Figure 14 is a graph of a direct test of the / 3-lamella form of the A4 / 3 (1-40); Figure 15 is a graph showing the ratio of denatured to native A4 / 3 (1-40); Figure 16 shows the conversion of SHaPrP90-231 from the α-helical conformation to the / β-lamella, during incubation at 37 ° C for 72 hours, as determined by circular dichromatism (CD) spectroscopy. The protein concentration was 5 mg / milliliter; Figure 17 shows the conversion of SHaPrP90-231 recombinant from the ot-helical to the ß-lamella conformation, during incubation at 37 ° C for 72 hours, as determined by direct differential assay. The increased signal of the native conformation at -24 hours indicates the destabilization of the native structure with the most open conformation followed by the conversion to the secondary structure (see Figure 16). The protein concentration was 5 mg / milliliter; Figure 18 shows that both HFIP and gricerol can avoid the conformational transition of a-a.-ß from recombinant SHaPrP90-231. The changes in the TRF signal are expressed as the fractional change, where the positive value indicates stabilization and the negative, destabilization. The experimental conditions were as in Figures 16 and 17; Figure 19: Pentosan polysulfate stabilizes the native conformation of SHaPrP90-231 at low concentrations; Congo red has no effect on the stability of SHaPrP90-231. The changes in the TRF signal are expressed as a fractional change, where the positive value indicates stabilization and the negative, destabilization. The experimental conditions were as in Figures 16 and 17; Figure 20: Z iterionic detergent ZW33-12 destabilized the native conformation of the a-helical Sha90-231. The experimental conditions were as in Figures 16 and 17; the changes in the TRF signal are expressed as fractional change, where the positive value indicates stabilization and the negative, destabilization; Figure 21 shows the calibration of a direct assay with purified human TTR in the normal conformation. Plates were developed with the anti-TTR primary antibodies (Accurate Chemical and Scientific Corporation, Westbury, NY) and anti-rabbit antibodies labeled with Eu. The data points and bars represent the average + SEM that was obtained from the four independent measurements; Figure 22 shows the calibration of a direct assay with purified human TTR in the amyloid conformation. Plates were developed with the primary antibodies of the anti-TTR and secondary anti-rabbit antibodies labeled with Eu. The data points and bars represent the mean ± SEM that was obtained from the four independent measures; Figure 23 shows the ratio between the denatured and native TTR signals in the normal and amyloid conformations, which were developed as described above. The data points and bars represent the mean ± SEM that was obtained from the four independent measures; Figure 24: The modified formula to calculate the amount of the TTR in the amyloid conformation from the data that was obtained through the direct test with the polyclonal antibody of the anti-TTR. The changes in the general equation reflect the inverse proportion of the denatured and native states for the normal and amyloid forms of the TTR. The difference between the fluorescence of the denatured state of the sample and that which was expected for the transition from the native normal protein to the denatured state is proportional to the amount of the TTR in the amyloid conformation. Fn - total signal of the native conformation; F ^ and F ^ - the signals of the normal and amyloid native conformations, respectively; Fd - total signal of the TTR in the denatured state; F ^ and FdA are the signals of the denatured normal or amyloid states of the TTR; ? Fn?, D - the total increase of the signal in the transition from the native to denatured states; ? FNn? D - increase in the normal conformation signal in the transition from the native to denatured state; fNn? d - correlation factor for the transition from the native to denatured state of the normal TTR; Figure 25 is a graph of the "prion index" (which is the ratio of antibody binding to denatured compared to the native PrP protein), compared to the concentration of the PrPSc in μg / milliliter. The results shown represent the mean ± SEM that was obtained from three different brains of Syrian LVG / LAK hamsters infected with different prion strains.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Before the present assays and methods are disclosed and described, it should be understood that this invention is not limited to antibodies, proteins, labels, assays or particular methods, since these could, of course, vary . It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, 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 that are used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference, to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed here are provided only by the description prior to the date of presentation of the present application. Nothing herein should be construed as an admission that the present invention is not authorized to set a date prior to this publication by virtue of the foregoing invention. In addition, the dates of the publication that is provided are subject to change if it is found that the current publication date is different from the one provided here.

DEFINITIONS The term "protein", as used herein, is intended to encompass any amino acid sequence and to include modified sequences, such as glycoproteins. The term includes proteins and peptides that occur naturally, as well as those that are synthesized recombinantly or synthetically. As used in connection with the present invention, it is specifically intended that the term "protein" cover naturally occurring proteins, which occur in at least two different conformations, wherein the two conformations have the same or substantially the same sequence of amino acids, but they have three different dimensional structures. 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-pathogen state. A specific and preferred example of a protein as used in connection with the description, is a PrP protein which includes the disease-free form referred to as the PrPc form, and the disease form referred to as the PrPSc. Although a prion protein of the PrPSc form of a PrP protein is infectious and pathogenic, the disease conformation of other proteins is not infectious, even if pathogenic. As used herein, the term "pathogen" may mean that the protein actually causes the disease or may simply mean that the protein is associated with the disease and therefore, is present when the disease is present. Thus, a pathogenic protein as used in connection with this description is not necessarily a protein that is the specific causative agent of a disease. The terms "treating", "treatment" and the like, are used interchangeably herein to describe a process by which a sample or portion thereof and specifically the proteins in the sample are physically and / or chemically manipulated. Thus, the proteins in the sample in a conformation related to diseases are caused to change to a different conformation with a higher binding affinity, with a binding pattern such as an antibody. Reference is also made to proteins treated as denatured or partially denatured proteins in a relaxed conformation, conformation that increases the binding affinity of the protein to a binding pattern such as an antibody. The treatment includes subjecting the sample to heat, pressure and / or chemicals. In a preferred embodiment, samples containing the PrPSc (which is the conformation related to diseases comprising the structural configurations of β-lamellae) are treated so that the protein assumes a different conformation (eg, comprising a -helical and / or a random coil configuration), which has four times or more affinity for higher antibody binding. The terms "PrP protein", "prP" and the like, are used interchangeably herein and should mean both the infectious particle form PrPSc of which it is known to cause diseases (spongiform encephalopathies) in humans and animals, and the non-infectious PrPc form which, under the appropriate conditions, becomes the infectious PrPSc form We use the terms "prion", "prion protein", and "prpSc protein" and the like interchangeably herein to reference to the PrPSc infectious form of PrP, and is a contraction of the words "protein" and "infection." The particles comprise mostly, if not exclusively, PrPSc molecules encoded by a PrP gene.Prions are different from bacteria, viruses and viroids, known prions infect animals to provoke them scraping, a transmissible, degenerative disease of the nervous system of sheep and goats, as well as bovine spongiform encephalopathy (BSE), or "mad cow disease", and feline spongiform encephalopathy of cats. The four prion diseases that are known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Streussler-Sheinker Disease (GSS), and ( 4) fatal familial insomnia (FFI). As used herein, the "prion" includes all forms of prions that cause all or any of these diseases or others in any animal that is used - and in particular in humans and in domesticated farm animals. The term "PrP gene" is used herein to describe genetic material that expresses proteins that include known pathogenic mutations and polymorphisms. The term "PrP gene" generally refers to any gene of any species, which encodes any form of a prion protein. In Gabriel et al, Proc. Natl. Acad. Sci. USA 89: 9097-9101 (1992), and in U.S. Patent No. 5,565,186, and in WO97 / 04814, which are incorporated herein by reference to disclose and describe these sequences, are described some PrP sequences that are commonly known. The PrP gene can be from any animal, including the "host" and "test" animals described in present and any other polymorphisms and mutations thereof, recognizing that the terms include others, such as the PrP genes that are not yet discovered. The protein that is expressed by this gene can assume a form either PrPc (no disease) or a PrPSc (disease). The term "antibody" represents an immunoglobulin protein, which can bind an antigen. It is intended that antibody, as used herein, include a complete antibody, as well as antibody fragments (e.g. F (ab), Fab, Fv) that can bind the epitope, antigen or antigenic fragment of interest. Preferred antibodies for the assays of the invention are immunoreactive or immunospecific and, therefore, bind specifically and selectively to a protein of interest, for example, an A4 / 3 amyloid protein or a PrP protein. Antibodies that are immunoreactive and immunospecific to both the native form without disease, and to the disease form treated, but not to the untreated form of disease (e.g., both for the native PrPc and the treated PrPSc, but not for for the non-native PrPSc). Antibodies to PrP are preferably immunospecific - for example, they are not substantially cross-reactive with related materials. In the published PCT application WO97 / 10505, which is incorporated herein by reference to reveal and describe antibodies, some antibodies are described specific that can be used in connection with the invention. This published PCT application corresponds to USSN 08 / 713,939, which is also incorporated herein by reference. The antibodies described in the PCT application, which selectively select PrPSc, should not be used in the present invention. The term "antibody" encompasses all types of antibodies, for example, polyclonal, monoclonal, and those that are produced by the phage display methodology. Antibodies of the invention that are particularly preferred are antibodies that have a relatively high degree of affinity for both the native PrPc and the treated PrPSc, but a "relatively low or substantially no binding affinity for the PrPSc. More specifically, the antibodies of the invention preferably have four times or more, more preferably fifteen times or more, and still more preferably 30 times or more binding affinity for both the native PrPc, and the prpSc. denatured, compared to binding affinity for native PrPSc. "Purified antibody" refers to one that is sufficiently free from other proteins, carbohydrates, and lipids with which it is naturally associated. preference "to the conformation of treated or denatured disease of a protein, such as the 3-lamella conformation of the A43 or the PrPSc protein (or a antigenic fragment thereof), and does not recognize or bind substantially to other non-related molecules in an antigenic manner. A purified antibody of the invention is preferably immunoreactive with an immunospecificity for a specific and more preferred species, immunospecific for native PrPc and for the treated or denatured forms of PrPc and PrPSc, but not for native or untreated PrPSc . "Antigenic fragment" of a protein (e.g., a PrP protein) means a portion of that protein, which can bind an antibody. By "specifically fixed" is meant high strength and / or high binding affinity of an antibody to a specific polypeptide, for example, the epitope of a protein, for example, a PrPc or A4 / 3 protein. The binding of the antibody to this epitope in its specific polypeptide is preferably stronger than the binding of the same antibody to any other epitope, particularly those that may be present in the molecules in association with, or in the same sample as, the polypeptide specific of interest, for example, binds more strongly to the epitope fragments of a protein such as PrPSc, so that by adjusting the binding conditions, the antibody binds almost exclusively to a site or fragments of the epitope of a desired protein, such as an epitope fragment that is exposes by treating the PrPSc and is not exposed in the untreated native PrPS. 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), which has A detectable label that can be detected The detectable label is normally linked by chemical fusion, but where the label is a polypeptide, it can be linked alternatively by genetic engineering techniques. Methods for the production of labeled proteins that can be detected are well known in the art. The detectable labels known in the art are usually radioisotopes, fluoroporos, paramagnetic labels, enzymes, etc. (e.g., horseradish peroxidase), or other fractions or compounds that emit either a signal that can be detected. (for example, radioactivity, fluorescence, color), or that emit a signal that can be detected after exposure of the label to its substrate. Various detectable label / substrate pairs (eg, horseradish peroxidase / diaminobenzidine, avidin / streptavidin, luciferase / luciferin), methods for labeling antibodies, and methods for using labeled antibodies are known in the art (see , for example, Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY)). The europium is a particularly preferred label. Abbreviations that are included herein include (for its acronym in English): CNS for central nervous system; BSE for bovine spongiform encephalopathy CJD for Creutzfeldt-Jakob disease; FFI for fatal familial insomnia; GSS for Gerstmann-Streussler-Sheinker Disease; Hu for human; HuPrP for human prion protein; Mo for mouse; Mo PrP for «mouse prion protein; SHa for a Syrian hamster; SHaPrP for prion protein from a Syrian hamster; Tg for transgenic; Tg (SHaPrP) for a transgenic mouse containing the PrP gene of a Syrian hamster; Tg (HuPrP) for transgenic mice that contain the complete human PrP gene; Tg (ShePrp) for transgenic mice that contain the complete sheep PrP gene; Tg (BovPrP) for transgenic mice containing the full cow PrP gene; prpsc for ia. prion protein scraping isoform; PrPc for the common, normal cellular isoform contained in the prion protein; ~ MoPrPSc for the scrap isoform of the mouse prion protein; MHu2M for a chimeric / human mouse PrP gene, where a region of the mouse PrP gene is replaced by a corresponding human sequence, which differs from the mouse PrP at codons 9: Tg mice (MHu2M) are transgenic mice of the invention, which include the chimeric MHu2M gene; MHu2MPrPSc for the scrap isoform of the human / mouse chimeric PrP gene; prpCJD for] _a CJD isoform of a PrP protein; Prnp0'0 for the ablation of the two alleles of an endogenous prion protein gene, for example, the MoPrP gene; Tg (SHaPrP + / °) 8l / Prnp0, ° for a particular line (81) of transgenic mice expressing SHaPrP, + / 0 indicates that it is heterozygous; Tg (HuPrP) / Prnp ° / ° for a hybrid mouse that was obtained by cross-linking a mouse with a human prion protein gene (HuPrP) with a mouse with the two alleles of the broken endogenous prion protein gene; Tg (MHu2M) / Prnp0 ° for a hybrid mouse that was obtained by crossing a mouse with a chimeric prion protein gene (MHu2M) with a mouse with the two alleles of the broken endogenous prion protein gene; TTR for transthyretin; FVB for a standard inbred strain of mice that is frequently used in the production of transgenic mice, since the eggs of FVB mice are relatively large and tolerate relatively well the microinjection of exogenous DNA; [PrPβ] - concentration of prion protein in the conformation / ^ - lamella; [ßAéß] - concentration of / 3A4 in the β-lamella conformation; [DRC] - concentration of a conformation related to diseases of a protein.

GENERAL ASPECTS OF THE INVENTION The test method comprises providing a sample suspected of containing a protein that assumes a first conformation and a second conformation related to diseases and that can detect a disease conformation of the protein when present in a very low concentration, in relation to the concentration of the conformation without disease. The first portion is preferably fixed to the surface of the solid support and thereafter contacted with a labeled antibody. He The antibody is of a type that binds to the protein in its first configuration, with a higher degree of affinity (four times or more) than with that which binds to the protein in its second conformation related to diseases. The second portion of the sample is then treated in a manner that causes any protein in the second disease-related conformation to assume a different conformation, conformation having a higher affinity degree (four times or more) for the antibody labeling, compared to the affinity for the protein in the second conformation related to untreated diseases. After, preferably the second untreated portion is fixed to the surface of the solid support. Then, the treated protein bound to the support is contacted with a labeled antibody, under conditions that allow the antibody to bind to the proteins in the first configuration of the proteins in the different configuration they assumed. After. that antibodies labeled with sufficient time, temperature and chemical conditions (eg, pH) have been provided to bind to the appropriate proteins in the respective portions, the level of binding of the labeled antibody to the protein in each portion is determined. A highly sensitive assay is used, such as an assay that includes time resolved, enhanced dissociation fluorescence, making it possible to detect concentrations in an amount in the range of about 1 x 103 particles per milliliter or less. A high degree of sensitivity is required, because the concentration of protein in the conformation of disease will be very low in most samples, compared to the concentration of the protein in the disease-free conformation, for example, 3 orders of magnitude or more of difference. For example, the disease-free conformation of the protein may be present in an amount of about 1 x 108 particles / milliliter, while the protein conformation of the protein is present only in an amount of 1 x 104 particles / milliliter. In this way, any increase in the signal that is noted because the conformation of disease of the protein is treated, will be very small in relation to the signal that is being obtained from the protein in the conformation without disease. After the level of fixation for the two portions of the sample is obtained, the levels are compared. For example, the level of binding of the labeled antibody to a protein in the first portion is subtracted from the level of binding of the antibody to a protein in the second portion. The difference between the two reflects the amount of protein present in the original sample that was in the second conformation related to diseases - after adjusting for differences (if any) that were caused by the increase in the binding affinity of the protein in the first portion. More specifically, there may be some differences with some proteins due to the effect of the treatment on the proteins that are in the native conformation without disease - for example, the treatment exposes more epitopes of the protein in the conformation related to diseases. This differential should be considered when reaching conclusions regarding whether the original sample included proteins in the second conformation related to diseases. Within the Figures 3 and 4 the consideration for this effect is shown. A comparison of antibody binding to an untreated sample, which contains only the native protein in its disease-free configuration with the same native protein after treatment, is shown in Figure 3. As shown in Figure 3, there is some difference between the results that were obtained with the treated protein that shows a stronger signal, in that the treatment increased the affinity of binding of the protein. However, Figure 4 shows the same results when the original sample included proteins that were in the second conformation diseases. Native proteins that are not treated provide a very weak signal. However, the treated proteins provide a very strong signal. The large differential enters the treated samples and does not treated, it is a clear indication that the original sample included proteins Gon the second conformation related to diseases, in which these proteins do not bind to antibodies or bind to antibodies with a very low degree of binding affinity. However, after treatment, these proteins bind to the antibodies as well or almost as well or better than the proteins in the disease-free conformation that were treated. The assay can be used to detect the presence of the disease conformation of a given protein within • any type of sample. Some of the most typical samples to be tested include pharmaceutical products that include components that are derived from living mammals or that use materials that are derived from living mammals in their processing. It would also be desirable to test organs for transplantation and food items such as beef suspected of containing infectious prions. The invention could also be used to test for the presence of the disease conformation of one or more types of proteins, such as infectious PrPSc in pharmaceutical, cosmetic, biopsy or autopsy tissue, brain, spinal cord, peripheral nerve. , muscle, cerebrospinal fluid, blood and blood components, lymph nodes, and cultures derived from animals or humans infected or potentially infected by the "forms of protein disease, such as prions TREATMENT OR DENATURALIZATION As indicated above, the "treatment" may include the exposure of the proteins to any physical and / or chemical means that cause the protein, which is originally present in a disease-related conformation, tightened, assume a more conformational conformation. relaxed that has a higher degree of binding affinity for any binding partner, such as antibodies. In general, treatment or denaturation, as it is sometimes called in the present, includes subjecting the protein to some means that cause the epitopes in the protein, which had not been previously exposed, to be exposed in such a way that an antibody or any other fixation partner can be fixed to the epitope that has just been exposed. The treatment methods that may be used include: (1) physical, such as pressure or hydrostatic temperature, (2) chemical, such as acidic or line pH, salts-chaotropic, denaturing detergents, and proteinases such as Proteinase K, and (3) combinations of the above. Treatment time will vary depending on the treatment used, but should be done long enough to expose new fixation sites, but not so long as to completely denature the protein.

PREVIOUS TREATMENT OR REMOVAL / DESTRUCTION OF THE PROTEIN Before carrying out the treatment or the antibody test of any portion of the sample, it may be desirable to subject the sample to a previous treatment. The previous treatment is carried out with the objective of destroying or removing the disease-free form of the protein present in the sample. Examples of the pre-treatment methodology include producing a column that includes fixed antibodies to the support surfaces, which antibodies bind to the disease-free conformation of the protein, thereby removing as much of the conformation without disease from the protein as possible. proteins. Alternatively, the sample may be subjected to physical treatment such as pressure or hydrostatic temperature alone or in combination with chemicals, such as acids or lines as indicated above in the "treatment" phase, but performed over a period of time more extensive and by way of destroying the proteins present in the sample, which proteins are in the conformation without disease. In some cases, proteins will be destroyed in the conformation without disease and disease. However, a greater relative percentage of the proteins in the disease-free conformation will be destroyed because these proteins are initially in a more conformation. loose, which is more vulnerable to destruction. In this way, the pretreatment methodology results in a sample that includes a relatively lower concentration of the disease-free conformation of the protein relative to the concentration of disease conformation of the protein. This increases the sensitivity of the assay, making it possible to detect lower concentrations of the disease conformation of the protein. The removal of the proteins on the destruction of these is preferred, because the destruction will diminish the sensitivity if the conformation of the disease is destroyed. In our lawyer registration number 6510/098001 of the patent application, which was filed on the same date as the present application entitled "Process for Concentrating Protein with Disease Related Conformation", a particularly useful pretreatment method is described. .

FIXING PROTEINS TO SUPPORT SURFACES The methods of chemical or affinity coupling of the PrP protein to the plastic support are 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 the denatured form of the PrPSc with, preferably, at least 4 times less reactivity with the infectious PrPSc, assuming the same amount of the antigen.

USING THE TEST TO DETECT PRIONS 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 e-j_ sample material or in the brains of susceptible animals that were injected with this material. The sample is divided into two aliquots. The first aliquot is cross-linked to the solid plastic support in the native conformation through a chemical activation step under non-denaturing conditions, ie, without treatment. First the second portion of the sample is treated and then reticulated to the plastic support. The two portions of the sample material react in situ with the labeled antibodies which preferably recognize the native PrPc or the PrPSc treated from the given animal species. The amount of the antibody bound to the treated or native conformations of the PrP protein is recorded by the signal of the IgG tag. The excess of the signal that was obtained with the denatured sample on that which was expected from an increase in the signal obtained with the native a-helical conformation of the PrPc (that is, the increase over the adjusted amount) , is the measure of the amount of PrPSc structured 3-lamella infectious in the original sample. In the formulas provided herein, the formula that was developed to calculate the content of the PrPSc is shown and exemplified in Example 11. The diagnosis of the prion disease is established by three procedures: (1) measurement of the sample treated alone and by detecting the increase in the total amount of PrP in the sample that was previously examined on the background levels of PrPc that were obtained from the normal controls; (2) calculation of the ratio between the denatured against the negative signal for the given antibodies - for example, values higher than 2.2 for the 3F4 IgG labeled with Europium, indicates the presence of the PrPSc, preferably using time-resolved fluorescence, of improved dissociation; (3) 'evaluation of the signal excess of the denatured sample on which is expected from the increase in the signal for the a-helical conformation of the PrPc, as a measure of the amount of the structured PrPc of / 3-lamella infectious in the original sample. Preferably, before step (1), the method uses a pre-treatment step by means of which the PrPc is removed or destroyed in relative amounts greater than that of the PrPSc. In a Syriac hamster brain infected with scraping, the concentration of the PrPSc is 5-10 times more higher than PrPc when animals get sick. At this time, the prion title in their brains is 107 - 108 ID50 units / milliliter of 10 percent homogenate. The highly quantitative system that we have developed allows us to subtract the PrPc signal. Subtraction can be easily performed when the concentration of the PrPSc is greater than the PrPc. However, subtraction becomes difficult when the concentration of the PrPSc is much lower than the PrPc. To give attention to the above problem, the present assay uses the principle of affinity between the different conformations of the antigen and the antibody. To measure the concentration of the PrPSc when it is much less than the PrPc, the detection system must have an extreme sensitivity and a linear range of at least 104. The assay described here can easily detect the PrPSc at a concentration of (approximately) 50 pg / milliliter, using the IgG labeled with Europium. Assuming 105-106 of PrPSc molecules per unit of ID50, the present assay can easily detect 5 x 102 - 5 x 103 ID5o units per milliliter. The assay can detect PrPSc in mixtures (by the direct method), wherein the concentration of PrPSc is less than 1 percent of the concentration of PrPc. Additional sensitivity can be achieved by immunoprecipitation, using a sandwich format for a status test solid, differential centrifugation with detergent extraction to remove the PrPc, the transgenic animal method or combinations of these methods. A conservative calculation is that these procedures should allow the measurement between 5 and 50 units of ID50 per milliliter or less conservatively, to measure between 0.1 and 0.01 units of ID50 per milliliter. These measures will provide rapid, "positive" means to establish biological sterility, which is the "absence" of infectivity.

METHOD OF IDENTIFICATION OF MEDICATION The tests described above, which can identify the presence of a conformation related to diseases of a protein, can be applied to identify the compounds that could then be used as drugs to treat or prevent diseases associated with the conformation of protein disease. For example, compositions of which "known to include conformations related to diseases of a protein, for inoculating a transgenic mouse of the type disclosed and described in U.S. Patent No. 5,565,186 and PCT publication WO97 / 04814. Mice that were inoculated with a compound that is believed to prevent infection can then be treated after providing a sufficient period of time for the Incubation, the mouse brain is removed and tested using the assay described above, to determine if the compound of the treatment was effective in preventing infection. The methodology could also be applied to situations where the infection has already taken place, with the aim of determining whether a particular compound could stabilize the infection, that is, avoid further formation of the protein's disease conformation. The assay of the present invention can be used in combination with the methodology described in USSN 08 / 556,823 issued above, which was filed on November 2, 1995, which is incorporated herein by reference. This application describes the compounds that can be contacted with the proteins in a conformation without disease, with the aim of converting these compounds to a conformation of disease. The methodology is useful because it allows the compounds to be traced by their ability to prevent formation of the conformation related to diseases of the protein. The assay of the present invention makes it possible to accurately measure the effect of any test compound on preventing the formation of the disease-related conformation. Based on the foregoing, it can be observed that the invention includes a method of screening compounds, which affect the conformational form of any protein. such as the PrP protein having a first related conformation without disease (e.g., PrPc) and a second conformation related to diseases (e.g., PrPSc). The method includes first, providing a sample having the protein present in the first conformation without disease. The sample is then contacted with a test compound, which is being screened for its therapeutic utility. Then, after adding the test compound, the sample is contacted with a compound or group of compounds that induce the protein in the first conformation without disease, to convert it to the second disease conformation. After allowing a sufficient period of time, the sample is tested using the present invention. The assay of the present invention will make it possible to determine exactly how the test compound affected the conversion of the protein from the disease-free conformation to the disease conformation. It will be evident to those skilled in the art to read this description, that the test compound can be added at different points in time, in relation to the addition of the compound that affects the conformational change. By the same, the methodology can be used to determine the ability of a test compound to stabilize additional changes from disease-free conformation to disease conformation and / or to determine the ability of the test compound to avoid start of any conversion from the conformation without disease to the conformation of disease. Although USSN 08/556, 823 'discloses a means for converting a disease-free conformation to a disease conformation, other means of obtaining the same result will be apparent to those skilled in the art, after reading the present application and reviewing the condition of the technique in connection with it. In accordance with the foregoing, it is intended that the present invention encompass any physical, chemical or biological medium that could be used to convert a protein within a first conformation without disease, to a second conformation of disease and apply the assay described above. . Furthermore, it is intended that the present invention encompass therapeutic compounds that are obtained as a result of performing the screening method of the invention, ie, the compounds that are produced by the method of the invention.

- ANTIBODIES Methods for generating antibodies are generally known to those skilled in the art. Because the disease form is often in a tighter configuration than the disease-free form, with fewer epitopes exposed, one can easily generate antibodies that only bind to the disease-free form of the protein or wing. disease form treated. For example, antibodies that detect the treated forms of the Prpsc protein and of the PrPc protein can be created by means of immunizing rabbits or mice with a-helical conformations or recombinant PrP, native PrPc from animal brains, synthetic peptides in conformations. a-helical or random coil, or against denatured PrPSc or PrP 27-30. Only antibodies with an affinity of at least 4 times greater for PrPc (or the denatured conformation of PrPSc from the same species) should be selected, in comparison with their affinity for PrPSc. The antibody generation, purification, labeling and detection method may vary. The IgG or Fab can be purified from different sources, by affinity HPLC using protein exclusion HPLC, column A and size. Purified antibodies can be labeled with Europium and detected by time-resolved fluorescence. The antibody that binds to different conformations of the PrP protein can be measured by resolved fluorescence by time, of enhanced dissociation. However, the IgG detection system fixed to PrP on the solid support in situ or in solution may vary. In addition, it is possible to use direct or indirect immunological methods, including direct radiolabels, fluorescence, luminescence, avidin-biotin amplification, or enzyme linked assays with colored or luminescent substrates. In the United States Patent Number 4,806,627, issued on February 21, 1989, which describes the monoclonal antibody 263K 3F4, which is produced by the cell line ATCC HB9222 deposited on October 8, 1986, which is incorporated in The present invention, as reference, describes an antibody that can be used in the invention. The cell line producing the antibody can be obtained with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852. In general, the scraping infection can not produce an immune response, with the host organisms being tolerant to the PrPSc from other species. In WO97 / 10505, which was published on March 20, 1997, antibodies are described that are already fixed to PrPc or PrPSc. Any antibody that binds to PrPc and not to PrPSc can be used, and those skilled in the art can generate these known processes of use, for example, see methods for producing page display antibody libraries in the US Patent. United States of America Number 5,223,409. However, polyclonal anti-PrP antibodies have been raised in rabbits, after immunization with large amounts of formic acid or SHaPrP 27-30 denatured with SDS [Bendheim, Barry et al. (1984) Nature 310: 418-421; Bode, Pocchiari et al. (1985) J Gen Virol 66: 2471-2478; Safar, Ceroni et al. (1990) Neurology 40: 513-5171. 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 et al. (1987) J Virol 61: 3688-36931. These antibodies were generated in comparison with formic acid or PrP 27-30 denatured with SDS and are. capable of recognizing native PrPc and treated or denatured PrPSc from SHa and humans equally well, but do not bind to PrPMo. Not surprisingly, the epitopes of these antibodies are mapped to regions of the sequence that contain the amino acid differences between SHa- and PrPMo [Rogers, Yehiely et al. (1993) Proc Natl Acad Sci USA 90.:3182-3186]. It is not completely clear why many antibodies of the type described in the publications cited above will be bound to PrPc and treated or denatured PrPSc, but not to native PrPSc. Without being bound by any particular theory, it is suggested that this should take place because the epitopes that are exposed when the protein is in the PrPc conformation are not exposed or are partially hidden in the PrPSc configuration - where the protein is relatively insoluble and It is folded together more compactly.

For purposes of the invention, an indication that no fixation occurs means that the equilibrium or affinity constant Ka is 106 1 / mol or less. In addition, the fixation will be recognized as existing when the Ka is at 107 1 / mol or more, preferably 108 1 / mol or more. The binding affinity of 107 1 / mol or more may be due to (a) a single monoclonal antibody (ie, large numbers of a class of antibodies) or (2) a plurality of different monoclonal antibodies (eg, 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). Preferred selected antibodies will be fixed at least 4 times more avidly to the treated or denatured prpSc forms of the protein when compared to their binding to the native conformation of PrPSc. The fourfold difference in binding affinity can be achieved by using many different antibodies according to (1) - (3) above, and as such some of the antibodies in a mixture can have less than a fourfold difference. A variety of different types of assays of the invention can be used with one or more different antibodies. Those of skill in the art will recognize that antibodies can be labeled with known labels, and used with currently available robotics, assays of sandwich, electronic detectors, flow cytometry, and the like.

QUANTITATIVE CALCULATIONS With the use of the methodology described above it is possible to calculate the difference between the amount of signal obtained from a sample that has not been treated, and the signal obtained with a sample that has been treated. This difference represents (after adjustment for the effect of the treatment on the non-disease conformation) the amount (concentration) of protein in the disease conformation present in the original sample. After obtaining the difference, the formula given below can be used to calculate the amount of protein in the disease conformation present in the original sample per unit volume. a > Fn = Fna + Fn / 3? Fna = Fn "Fnß 'Fnß ~ fon¿ ° b) Fd = Fcta + Fdβ? Fn? D =? Fan? D +? F / Jn? D? E n? D =? Fd" Fn -? Fcm? D [? PrP ^] or [DRC] _ AFßn? D = Fd - (Fn *? Fan? D) The definition of each of the previous variables is provided below. F - fluorescence signal (note that it can be used any detectable signal); Fn - fluorescence signal of the native conformation; Fna and Fnβ - fluorescence signals of the a-helical and native 8-lamella conformations, respectively; Fd - fluorescence signal of PrP in the treated or denatured state; Fda Y Fdß ~ are the signals of the CePecoidal or denatured PrP; ? Fn? D - increase of the fluorescence signal in the transition from native to denatured state; ? Fcm? D ~ increase in the fluorescence signal of the a-helical conformation in the transition from the native to the denatured state; ? F / 3n? D ~ increase in the signal of the conformation of (S-lamellae in the transition from the native state to the denatured one; fcyn? D - correlation factor for the transition from the native to the denatured state of the PrP ^ -helical; [PrPo] - concentration of prion protein in the conformation of / S-lamella. [DRC] - concentration of any protein in the disease-related conformation.The formula provided above is used to specifically calculate the concentration of the prion protein in the conformation of / 3-lamella. However, the same formulas can be used to calculate the concentration of any protein, i.e. the concentration of any reduced disease conformation of a protein such as [ßA4β]. More generally, [DCR] represents the concentration of conformation related to disease of a protein. To provide a specific example, the foregoing definitions have been provided specifically with respect to -PrP proteins, proteins that include at least one non-disease relaxed conformation (PrPc), including an a-helical configuration and at least one conformation (PrPSc ) reduced, related to disease, which includes a / 3-lamella configuration. The formulas are used to calculate the concentration of the conformation related to disease of the protein present in the sample. For the specific formulas and definitions provided above, the formulas are used to calculate the concentration of prion proteins that include the / 3-lamella configuration (see Example 11). The signal that was used to calculate the previous formula is a fluorescent signal. However, any signal that can be detected can be used. The total signal is represented by Fn, which is a combination of the signal received from the conformations related to disease and not disease.

This is a signal that would be calculated from the Number 1 portion, which is not treated according to the test described above. The variable Fd is the signal obtained by treating the Number 2 portion of the sample. This signal is a combination of the signal received from the protein treated in the non-disease conformation, plus the protein treated in the disease conformation. It has been recognized that there is a difference in the signal obtained by treating a sample that does not include any conformation related to protein disease. The difference must be taken into account to obtain an accurate reading. The difference in the signal obtained between the native sample and the treated sample is, of course, a combination of the difference in the signal obtained by treating the conformation related to disease, and the non-disease conformation. The increase in the signal obtained can be calculated by treating the disease conformation, ie, the difference between the signal of the untreated disease conformation, and the signal received from the conformation of the treated disease, by subtracting the signal received from the treatment of the entire sample of the signal received from the calculation of the increase in the signal obtained from the conformation not of untreated disease and the conformation of not treated disease. Using these equations it is possible to produce the final equation that provides the concentration of the protein in the disease conformation present in the original sample (see Example 11).

DIFFERENTIATION (TYPIFICATION) OF PROTEIN STRAINS Different animals, including humans, can be infected with different strains of pathogenic proteins. A "mutation table" is provided herein to list some of the different mutations associated with different strains of prion infections. Sometimes it may be important which specific strain has infected an individual, in the sense that that information may be useful to (1) provide a more accurate diagnosis, (2) administer the appropriate treatment, or (3) determine the source of the infection by comparing the strain with a strain in a probable source of infection. The particular pathogenic protein strain causing an infection can be determined from two pieces of information that can be calculated using the present invention. Example 11 shows how the present invention can be used to determine the absolute amount, ie, the concentration of prions in a sample of given size. Example 8 shows how to calculate the "prion index" which is the ratio of antibody binding to denatured PrP protein: native. A "prion index" for other proteins is the fixation ratio of any partner Fixation to the denatured form: native to the protein. In general terms, the "prion index" is simply a numerical characterization of the influence that treatment has on the protein. To determine the particular strain one must first calculate a standard for each strain. This is done by determining the influence (prion index) of a particular treatment on a known amount of a known strain. This can be delineated as shown in Figure 25. Once the standard is calculated, the strain of other samples can be quickly determined - the standards are preferably calculated at a number of different concentrations for each strain. To determine the strain of a simple sample, one calculates the protein concentration in the sample, and the influence of the treatment on that concentration. To determine the strain, the results are compared with the standard (as in Figure 25). Example 18 is a specific example thereof, taken in combination with Figure 25. The same concentration of the same strain will be made in the same manner by means of a given treatment. However, the same concentration of different strains will be made differently, by means of the same treatment, making it possible to determine the strain.

DISEASES ASSOCIATED WITH INSOLUBLE PROTEINS Much of the description and specific examples provided herein 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 for the determination of 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, A / 3 peptide, al-antichymotrypsin, tannin, non-Aß component Prion diseases, Creutzfeld Jakob disease, raspberry and bovine spongiform encephalopathy Prpsc ALS SOD and neurofilament Pick disease Pick body Parkinson's disease Lewy body Diabetes Type 1 Amiline Multiple myeloma-plasma cell dyscrasias IgGI chain Familial amyloid polyneuropathy Transthyretin Medullary thyroid carcinoma Procaleitonin Chronic renal failure / 32 - microglobulin Cardiac-congestive failure Atrial natriuretic factor Senile and systemic cardiac amyloidosis Transthyretin Chronic inflammation Amyloid of Serum A Arteroesclerosis ApoAl Familial amyloidosis Gelsolin It should be noted that the insoluble proteins listed above each include a number of variations or mutations that result in different strains that are all encompassed herein. Subsequently, the mutations and pathogenic polymorphisms known in the PrP gene related to prion diseases are given, and in the United States Patent Number 5,565,186, issued on October 15, 1996, the sequences of human, sheep, and bovine MUTATIONS TABLE Human Mutations Polymorphisms of Polymorphisms of Pathogenic Bovine Polymorphisms Human Sheep Insert of 2 Codon 129 Met / Val Codon 171 Arg / Glu 5 or 6 octarrepetitions octarrepeticiones Insert of 4 Codon 219 Glu / Lys Codon 136 W / Val octarrepeticiones insert of 5 octarrepeticiones insert of 6 octarepeats insert of 7 octerepetitions insert of 8 octerepetitions insert of 9 octerepetitions Codon 102 Pro-Leu Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Alto Csdón 175 Asp-Asn Coddn 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 also be noted that these proteins have two different three-dimensional conformations with the same amino acid sequence. One conformation is associated with disease characteristics and is generally insoluble, while the other conformation is not associated with disease characteristics and is soluble. The methodology of the present invention is not limited to diseases, proteins and strains listed.

DETECTING THE ß-LAMINILLA FORM OF ßA4 One aspect of the invention involves a two-step process for diagnosing Alzheimer's disease, based on the presence of a reduced form of a protein (amyloidosis per / 3A4), by quantitative measurement The ßA4 protein ß-form in the sample material, for example, in the brain or bodily fluids, is divided into two aliquots.The first aliquot is cross-linked to a , solid plastic support (long chain polymeric material) in the native conformation, through a chemical activation step, under the non-denaturing conditions. The second portion of the sample is first denatured, and then reticulated to the plastic support. Both portions of the sample material react in situ with the labeled antibodies which preferably recognize soluble ßAé or denatured / 3A4 of the human or of a given species of animal. The amount of antibody bound to the denatured or native conformations of the ßA4 protein is recorded by the labeled secondary antibody signal. The excess of the signal obtained with the denatured sample, in comparison with that expected change in the signal obtained with the native a-helical conformation of the ßA4 protein, is the measure of the amount of structured ßA4 as / 3-laminiÍla in the original sample. Previously, the formula developed for the calculation of the content of 3A4 was provided, in connection with the calculation of the content of PrPSc. The diagnosis of amyloidosis by 3A4 (disease of Alzheimer's) is established by three procedures: (1) the measurement of the denatured sample alone, and by detecting the increase in the total amount (concentration) of 3A4 in the sample examined on the background levels of the soluble SA4, obtained of normal controls; (2) the calculation of the ratio between the denatured signal against the native one for a given antibody (protein index) - for example values greater than 2 for the monoclonal antibody 6F3D and the secondary antibody labeled europium; (3) the evaluation of the signal change of the denatured sample on that expected signal change for the a-helical conformation of jSA4, as a measure of the amount of ßA4 structured as jS-infectious lamellae in the original sample. The formula developed for the calculation of the ßA4 content was previously provided. The particular strain of ßA4 can also be determined using the same methodology described above to determine the PrPSc strain in a sample. The invention provides a direct diagnostic method for detecting the presence of pathogenic forms of the protein / 3A4 in pharmaceutical products, biopsy tissue or autopsy, brain, spinal cord, peripheral nerves, muscle, cerebrospinal fluid, blood and blood components, lymph nodes, and in animal or human derived cultures that express or potentially express the ßA4 protein. The invention also makes it possible to follow the conformational transition of c¿-helical-to-α-lamella protein 3A4, or its fragments of synthetic or recombinant origin, and to provide a method for screening compounds for their ability to stabilize soluble conformation. normal of the ßA4 protein, and thus avoid conversion to the pathogenic ßA4 protein insoluble and structured as ß-lamellae. Typical methods of denaturation of the sample include: (1) physical, such as hydrostatic pressure or temperature, (2) chemical, such as acidic or alkaline pH, chaotropic salts, or denaturing detergents, and (3) combination of previous The methods of chemical or affinity coupling of the protein / 3A4 to a plastic support are described in the available literature, and these may vary. Antibodies used in the diagnostic assay can be polyclonal, monoclonal, or recombinant Fab, and must be specific species with preferential fixation to the soluble or denatured form of ßA4, preferably with at least a 2-fold difference in reactivity between ßA4 structured as a-helical and / 3-lamella, assuming same amount of antigen. The methods of binding 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, for the 'diagnostic test only. they should use the antibodies with an affinity of at least about 2 times for the denatured one, and compare with the conformation of native jS-lamella of ßA4 of the same species. Generation, purification, labeling and antibody detection methods may vary. The binding of the antibody to different conformations of the protein / 3A4 was measured by means of time-resolved fluorescence, of improved dissociation. However, the IgG detection system fixed to / 3A4 in solid support in situ or in solution may vary, and may use direct or indirect immunological methods, including direct radiolabels, fluorescence, luminescence, avidin-biotin amplification, or assays. linked to the enzyme with colored or luminescent substrates.

EXAMPLES The following examples are set forth in order to provide those of ordinary experience in the art a disclosure and description of how to make and use the assays of the present invention, and are not intended to limit the scope of what the inventors estimate as their invention, nor are they intended to represent or imply that the experiments below are all of, or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (for example, 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 the weight average molecular weight, the temperature is in degrees centigrade, and the pressure is at or near atmospheric.

EXAMPLE 1 EXPRESSION OF RECOMBINANT PRION PROTEINS For the development and calibration of the diagnostic assays, recombinant Syrian hamster prion proteins of the sequence 90-231 were re-folded to the a-helical or lamellar conformations as shown in FIG. describe [Mehlhorn, Groth et al. (1996) Biochemistry 3_5: 5528-5537]. The polymerase chain reaction (Perkin-Elmer) was used to amplify the DNA corresponding to different portions of the Syrian hamster prion protein, in order to bind it within E. coli vectors. HE synthesized many 5 'oligonucleotide primers with an Mlu I restriction site, within the C-terminal coding sequence of the STII signal peptide [Lee, Moseley et al. (1983) Infect Immun 42: 264-268; Picken, Mazaitis et al. (1983) Infect Immun 4_2: 269-275], and the initial amino acids of the appropriate PrP sequence. A 3 'oligonucleotide primer matching the 3' end of PrP, a stop codon and a Bam Hl restriction site, was used with each of the 5 'oligonucleotides. The amplified products were purified by the polymerase chain reaction, ligated into the previously digested vectors with Mlul / Bam Hl and transformed to DH5a. Clones containing the PrP insert were sequenced and transformed into the protease deficient expression strain 27C7 (ATCC # 55244). A large scale expression was performed, as described above, for other proteins, using a different medium [Cárter, Kelley et al. (1992) Biotechnoloqy 10: 163-137]; 500 milliliters of a culture grown overnight were inoculated in LB medium supplemented with ampicillin, into 7 liters of fermentation medium and an aerated (Braun, model E10) 10 liters. The cells were cultured at 37 ° C at a high agitation speed, and expression was induced. by starvation of phosphate. After 4 hours, a glucose solution was added at 50 percent, at a rate of 1 milliliter / minute; Glucose levels were inspected using a dipstick to measure the depth (Diastix, Miles Inc.) of glucose. A pH of 7.4 was maintained throughout the run, by automated addition of 10 percent H2SO4, or 24 percent NH40H. The final volume was 10 liters, in which an ODeoo of = 100 was achieved after 36 hours. E. coli was harvested by centrifugation at 10,000 x g for 30 minutes, and the resulting paste was stored at -20 ° C. For purification, 100 grams of E. coli paste were resuspended in 1 liter of 25 mM Tris-HCl, with a pH. 8.0, 5 mM EDTA (pH regulator A). This was centrifuged at 10,000 x g for 20 minutes, and the supernatant containing soluble periplasmic proteins was discarded. The pill was resuspended in 1 liter of pH regulator A, passed twice through a cell disruptor (Microfluidics International, model MF110), and centrifuged ,000 x g for 1 hour, after which the supernatant was discarded, and the pill was once washed in pH regulator A, and centrifuged again at 30,000 x g for 1 hour.

In this stage the pill can be stored at -20 ° C before another separation. This was subsequently solubilized in 8M of Gdn-HCl / 25 mM Tris-HCl, with a pH of 8.0 / 100 M DTT (pH regulator B), and centrifuged at 14,000 x g for 20 minutes, to remove the remaining insoluble matter. HE separated the 6 milliliter aliquots of the supernatant containing -200 milligrams of total protein, by size exclusion chromatography (SEC), using a HiLoad Superdex 200 column (Pharmacia) of 26 millimeters x 60 centimeters, levigating with 6M Gdn-HCl /12.5 mM Tris-HCl, with a pH of 8.0 / 5 mM DTT / l mM EDTA (pH regulator C) at a flow rate of 2 milliliters / minute. The fractions enriched by the recombinant prion protein were grouped as identified by sodium dodecylsulfate polyacrylamide gel electrophoresis, and further purified by reverse phase high performance liquid chromatography (RP-HPLC), using a C-4 column. (Vydac) of 25 millimeters x 25 centimeters; PH regulator 1: H20 / 0.1 percent TFA, pH regulator 2: acetonitrile / 0.09 percent TFA, flow rate of 5 mm / minute. The recombinant PrP protein was found in fractions containing 40 percent acetonitrile. If the levigated SEC was stored at 4 ° C for many days prior to reverse phase high performance liquid chromatography, the recombinant protein was levigated in previous fractions containing only 35 percent acetonitrile. The samples of the reduced protein and the refolded oxidized form were concentrated, using a Centricon column (Amicon) with a molecular weight cut.de 10,000 Da. The pH regulator for the reduced protein was 10 mM of MES, with a pH of 6.5, while the oxidized form was concentrated in the refolding pH regulator described above. The conformations of the refolded oxidized and reduced forms of SHaPrP90-231 protein were determined by circular dichromatism (CD) spectroscopy (Figure 1).

EXAMPLE 2 PURIFICATION OF NORMAL HMSTER PrPc AND PrPSc OF SCREED INFECTED H MSTER BRAINS Both proteins produced according to the Example were used 1 as standards for the prion assay, and to establish the sensitivity and range of linearity of the diagnostic method. Purified Syrian hamster brain PrPc was used for the calibration of prion protein detection, and was correlated with the results obtained in SHaPrP90-231 in the α-helical, β-lamellar, and denatured conformations. The PrPc protein was purified as described, with some minor modifications [Pan, Stahl et al. (1992) Protein Sci. 1: 1343-1352; Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90: 10962-109661. The protein content was determined by amino acid analysis. The purity of the PrPc protein, as demonstrated in the sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by silver staining and Western, was = 95 percent.

The standard Syrian hamster PrPSc was purified from the standard group of hamster brains infected with Sc237 scraping strain, as described, with only minor modifications [Turk, Teplow et al. (1998) Eur J Biochem 176: 21 -30]. The infectivity of this standard, as determined by an incubation time assay in Syrian hamsters, after intracerebral inoculation, was 107-3 ID 50 / milliliter, and a specific infectivity of 8.2 ID50 / milligram of PrPSc protein. However, the specific infectivity can vary from batch to batch ± 10 ° -5 ID50 / milligram. The protein content was determined by the BCA assay, using bovine serum albumin as a standard. The preparation was considered homogeneous with a larger band in the electrophoresis of sodium dodecylsulfate polyacrylamide gel, after silver staining and Western blots.

EXAMPLE 3 METHOD OF SELECTION, ETIOUETATION AND DETECTION OF THE ANTIBODIES USED IN THE TEST In other places, protocols and methods for the production and characterization of antibodies are generally described [Harlow and Lane (1988) supra: 726]. The data described in this and the following examples were generated with polyclonal antibodies N12 and P3 purified by immunoaffinity [Safar, Ceroni et al. (1990) Neuroloqy 40 .: 513-517; Rogers, Serban et al. (1991) J Immunol 147: 3568-3574], made against the synthetic peptides corresponding to the 90-145 (N12) and 222-231 (P3) sequence of Syrian hamster PrP [Barry, Vincent et al (1988) J Immunol 140 .: 1188-1193]; JS2 against PrP 27-30 from denatured Syrian Hamster [Safar, Ceroni et al. (1990) Neurolocrv 40: 513-517]. Elsewhere the development and characteristics of the monoclonal antibody 3F4 used in the assay are described [Kascsak, Rubenstein et al. (1987) J Virol 61: 3688-3693], and are described in the US Pat. Number 4,806,627, all of which are incorporated by reference to disclose and describe antibodies that can be used with the invention and methods for making those antibodies and related antibodies. More recently, denatured forms of recombinant Fab recognition of the prion protein were developed [Williamson, Peretz et al. (1996) Proc Natl Acad Sci USA 93: 7279-7282]. SHaPrP90-231 was covalently attached to the a-helical, 3-lamella and random coil conformations, to polystyrene plates activated by glutaraldehyde, and incubated with primary antibodies serially diluted. The amount of IgG that reacted with each conformation of SHaPrP90-231 was determined either directly with 3F4 IgG labeled with Eu, or indirectly with anti-rabbit or anti-mouse antibody labeled with europium, in accordance with the usual protocols, and the total signal was measured by fluorescence resolved by time, of enhanced dissociation. For the development of the assay, antibodies were selected with a proportion of denatured conformation signal against jS-lamella of SHaPrP90-231 equal to or greater than 4.

EXAMPLE 4 COMPETITIVE AND DIRECT TRIAL FORMAT Purified recombinant SHaPrP90-231 was diluted, refolded in an a-helical or ß-lamella conformation, in 5 percent (w / v) brain homogenate, obtained from PrP ° / ° of mouse and that did not contain any prion protein. The brain homogenate was made by three bursts of 30 seconds in a PowerGen homogenizer equipped with a disposable plastic probe in TBS, with a pH of 7.4, containing a cocktail of protease inhibitors (1 mM PMSF, 2 μg / milliliter of Aprotinin, and 2 μg / milliliter of leupeptin), and was rotated at 5 ° C for 5 minutes at 500 G in a tabletop centrifuge. The resulting supernatant was diluted 1: 1 in TBS with 4 percent final weight (volume / volume) of Sarcosyl, and homogenized again by three 30 second bursts in a PowerGen homogenizer. Then, the homogenate was added with different dilutions of SHaPrP90-231 in the a-helical or ß-lamella conformations. In a typical competitive assay, the PrP analyte in different conformations was previously incubated with 3F4 IgG labeled with europium, and then transferred to the polystyrene plate coated with SHaPrP90-231 in the denatured state by sodium dodecylsulfate. The results for the SHaPrP90-231 analyte in the a-helical and denatured state (Figure 2) indicate a marked difference in both available binding sites and the affinity of 3F4 IgG labeled with europium with different conformations of prion protein. In the direct assay, each sample of the dilution curve was divided into two aliquots: (1) untreated and designated native; (2) mixed with 4M final Gdn HCl, and heated for 5 minutes at 100 ° C and designated denatured. Both samples were diluted 20 times with H20, and the aliquots were loaded onto the polystyrene plate activated with glutaraldehyde. The plates, incubated overnight at 5 ° C, were blocked with TBS, at a pH of 7.8, containing 0.5 percent BSA (weight / volume) and 6 percent Sorbitol. (weight / volume). In the next step, these were washed three times with TBS, at a pH of 7.8, which contained 0.05 percent (volume / volume) of Tween® 20, and incubated with the europium-labeled antibodies listed above. The plates were developed after about 7 washing steps in the improvement solution provided by the Europium label supplier (Wallac Inc., Turku, Finland) and the signal was counted in the DELFIA 1234 Fluorometer (Wallac Inc., Turku, Finland).

EXAMPLE 5 DIFFERENTIAL TEST FOR DIFFERENT CONFORTS OF SHaPrP90-231 The parameters obtained from the direct assay with 3F4 IgG labeled with Europium were plotted as a function of the concentration. The results obtained with SHaPrP90-231 in the a-helical conformation (Figure 3) indicate a relatively small difference between the a-helical and denatured protein signal. The limit of sensitivity for denatured PrP in the presence of 5 percent brain homogenate is = l ng / milliliter, and the range of linearity over 3 orders of magnitude. In the SHAPrP90-231 lamellar-shape experiments (Figure 4), the 3F4 IgG labeled with europium was tightly bound to a denatured form of the protein. In contrast, reactivity with the native ß-lamella form of the protein only marginally exceeded the background, even at high concentrations of the protein. When the results were expressed as a ratio of the fluorescence of the denatured states against the native prion protein (Figures 3 and 4), the ratio for the ce-helical conformation is 1-1.8, and for the recombinant SHaPrP90-231 in the conformation of ß-lamella is 5-50. This effect was also used to analyze the PrP samples of unknown conformation, where the small increase in the 3F4 IgG signal labeled with europium, after the denaturation of PrP, is a characteristic of the ot-helical conformation. In contrast, the large increase in the signal on the expected change for the a-helical conformation is diagnostic of PrP90-231 in the conformation of ^ -laminilla (Figures 3 and 4). The results are expressed in two different ways: (1) as a proportion (Figure 6), where the index = 1.8 for the recombinant SHaPrP90-231 indicates that the protein was originally in all the "-helical conformation, and the >index; 1.8 indicates the presence of the ß-lamella conformation; (2) as a formula shown here, and exemplified in Example 11, wherein the increase in excess of the signal over that expected for an a-helical conformation is proportional to the amount of SHaPrP90- 231 in the conformation of 3-lamella.

EXAMPLE 6 QUANTITATIVE ESSAY FOR SHAPrP90-231 AND RECOMBINANT PrPc The input / output calibration for SHaPrP90-231 denatured in both the a-helical and jS-lamella conformations was linear within three orders of magnitude, and provides a high degree of confidence (Figure 5) for the trial. PrPc assays were also performed, diluted in series within the mouse homogenate of PrP0'0, which provided results with a high degree of confidence within the linearity range of 3 orders of magnitude. The assay was then calibrated by purified infectious PrPSc, in the presence of brain homogenate of 5% PrP0'0. The calibration with the PrPSc provided a linear response within a similar range. Using the differential method, the ratio of the denatured versus native brain PrPc signal was, for the 3F4 monoclonal IgG labeled with europium, of 2.2 (Figure 7), for polyclonal N12 and P3, of 1.0. The calibration for the PrPSc gave the proportion of >20 (Figure 8) for 3F4 IgG labeled with europium, and > 4 for antibodies N12 and P3. By using the formulas developed herein, the entire PrPc was in full range in an a-helical conformation. In contrast, the calculated amount of PrPSc in the infectious conformation of laminilla was as anticipated close to the total amount of prion protein.

EXAMPLE 7 DIAGNOSIS OF PRISON DISEASE BASED ON THE INCREMENT OF THE TOTAL PRION PROTEIN ON THE PHYSIOLOGICAL LEVELS OF PRPC The exact quantitative measurement of total PrP by exclusively evaluating the signal of the denatured sample gave an average value of PrPc in homogenates of Syrian hamster brain normal at 5 percent of 5.0 ± 0.2 μg / milliliter (Mean ± SEM) (Figure 9). Serial dilution of scraped-infected hamster brain homogenate (strain Sc237) within the mouse brain homogenate of PrP ° / ° gave total PrP values of 36.0 + 4 μg / milliliter (Figure 10), with ample linearity of the measurement. Because the only known condition for accumulation of prion protein is the accumulation of the infectious form of PrPSc, the increased levels of total PrP in the sample tested is indicative of the presence of prions. This prion assay can be used: (1) directly in the brain, tissue samples, body fluids or pharmaceuticals after the determination of normal control values; or (2) indirectly by detecting the elevation of total PrP in the brain of experimental animals inoculated intracerebrally with tissue, body fluid or pharmaceutical sample tested, which are suspected to contain prions.

EXAMPLE 8 DIAGNOSIS OF PRESSURE DISEASE BASED ON INCREASE IN SIGNAL PROPORTION BETWEEN DATATURALIZED PRP AGAINST NATIVE ("PRICE INDEX") the ratio between the affinity of the antibodies for the native hamster brain PrPc protein denatured against native is in the wide range of concentration for 3F4 IgG labeled with europium, between 0. 8-2.2. The ratio in the brain infected with scraping is through the range of complete linearity, on those values (Figure 11). The "prion index" gives a relative indicator of the presence of infectious PrPSc, and therefore of prions. This mode of prion testing is being used: (1) directly in the brain, tissue samples, body fluids or pharmaceuticals, after index determination for normal controls; or (2) indirectly by detecting the high index of denatured / native prion protein in the brain of experimental animals inoculated intracerebrally with tissue, body fluid or pharmaceutical sample tested, suspected of containing prions.

EXAMPLE 9 DIAGNOSIS OF PRESSURE DISEASE FROM DIFFERENTIAL TESTING THROUGH CALCULATION OF PrPSc CONTENT By using the direct assay and the formulas provided herein, there is no detectable amount of the ß-lamella form of PrPSc in the homogenate of normal brain. Conversely, the majority of the total PrP in the hamster brain infected by scraping was due to the accumulation of PrPSc (Figures 9 and 10). The correlation between the prion titer and the PrPSc calculated from the formula has a broad linearity and a sensitivity cut of ~ 103 ID50 / milliliter (Figure 11). The formula gives a quantitative indicator of the presence of the PrP protein in the abnormal conformation that correlates quantitatively with the prion titer. This mode of prion assay is used: (1) directly in the brain, tissue samples, body fluids or pharmaceuticals; or (2) indirectly by calculating the content of PrPSc in the brain of experimental animals inoculated intracerebrally with tissue, body fluid or tested pharmaceutical samples suspected of containing prions. After establishing a calibration curve between the PrPSc and the prion title, it is possible to estimate the titer directly by the PrPSc content.

EXAMPLE 10 MEASUREMENT OF THE CONVERSION OF A-HELICOIDAL TO B-LAMINILLA OF THE PrP IN VITRO PROTEIN TO TRACK THE GENERATION OF NEW PRESSES AND POTENTIAL DISEASE THERAPEUTICS The aliquots of a 100 μg / milliliter solution were incubated in the same way as a-helical of SHaPrP90-231, or the recombinant SHaPrP29-231, or the corresponding recombinant or synthetic peptides of the prion protein, in 20 mM Na pH buffer, with a pH of 5.5, for 24 hours at 37 ° C with 10"concentrations 3 • - 10"d M glycerol, cyclodextrins, heparin, heparin sulfate, Congo Red, cholesterol ester, dimyristoyl phosphatidylcholine. -The samples were then divided into two aliquots: (1) untreated, designated native; (2) mixed with 4M final Gdn HCl and - heated for 5 minutes at 100 ° C, designated denatured. Both samples were diluted 20 times with H20, and the aliquots were loaded onto the polystyrene plate activated with glutaraldehyde. The plates, incubated overnight at 5 ° C, were blocked with TBS, at a pH of 7.8, containing 0.5 percent BSA (weight / volume) and 6 percent Sorbitol (weight / volume). In the next step, these were washed three times with TBS, at a pH of 7.8, containing 0.05 percent (volume / volume) of Tween® 20, and incubated with the 3F4 IgG labeled with europium. The plates were developed after about 7 additional washing steps in the solution of improvement provided by the europium label supplier (Wallac Inc., Turku, Finland) and the signal was counted in the DELFIA 1234 Fluorometer (Wallac Inc., Turku, Finland). The degree of conversion of the a-helical conformation to the / 3-lamellae of the PrP was calculated from the "prion index" or alternatively from the formulas provided herein. Some compounds that inhibit conversion by apparently stabilizing the native-like conformation of the prion protein may have therapeutic potential in vivo.

EXAMPLE 11 The following example demonstrates the test method with the hamster brain homogenate from Syria infected with scraping, diluted 4 times in PrnP0'0 mouse brain homedod: a) Each plate was calibrated with an internal standard that consisted of five dilution points of SHaPrP90-231 denatured. The time resolved fluorescence (TRF) of total PrP was developed with the 3F4 IgG labeled with europium, and the values of the resolved fluorescence were plotted by time as a function of the concentration of PrP (Figure 4). The data is fit within a linear or polynomial equation, using the least squares method, and the best function for the PrP calculation is selected denatured PrP [μg / ml] = -0.22935 + 0.00026567 * [TRF] + 0.0000000012255 * [TRF] 2 (1) b) In the rest of the plate, the native and denatured aliquots of the hamster brain homogenate from Syria infected with scraping were incubated, diluted 4 times, and cross-linked with the plastic support, with 3F4 IgG labeled with europium. The total PrP content was calculated in accordance with the above formula, from the fluorescence signal of the denatured sample: c) The proportion of the fluorescence signals between the denatured and native samples was calculated: The normal value of PrPc determined from normal hamster brain homogenate is 2: 2; values over 2.2 are considered abnormal, and indicate the presence of PrPSs. d) The excess of the fluorescence signal on that expected for the a-helical PrP at the transition from the native to the denatured state is a measure of the amount of PrPSc, and is calculated in accordance with the formulas provided: ? F ß n? D (F -a n? D wherein f = is the maximum value of the factor for the fluorescence signal at the transition from the native to the denatured state of PrPc; Fd is the fluorescence of the denatured sample; and Fn is the fluorescence of the native sample. Then the amount of PrPc is calculated from? F ^ n? D and equation (1): The positive value calculated for the ß-lamella form of the prion protein indicates the presence of PrPSc.

EXAMPLE 12 EST NDARS OF THE SOLUBLE ("HELICOIDAL) AND INSOLUBLE (^ -LAMINILLA) FORMS OF THE PROTEIN &A4 Soluble forms of 3A4 (1-40) and ßA4 (1-42) were obtained with Bachem (Torrance, CA ). A portion of the fresh freeze-dried peptide, at a pH of 7.4, containing 20 percent (volume / volume) of HFIP (hexafluoroisopropanol; 1, 1, 1,3, 3,3-hexafluoro-2-propanol) was solubilized in PBS. or SDS (sodium dodecyl sulfate) at 1 percent (weight / volume) at the final protein concentration of 50μM; this protein was designated "soluble" and stored at -80 ° C until it was used. A second portion of the peptide was resuspended in PBS, at a pH of 7.4, at the final concentration of = 350 μM, and incubated for 72 hours at 37 ° C. This portion of the protein was designated "insoluble" and stored until used at -80 ° C. Both proteins were used as standards for the development of the assay, and to establish the sensitivity and range of linearity. CD spectroscopy (circular dichromatism) (Figure 12) showed that the soluble protein had an a-helical conformation (see the solid line in Figure 12). In contrast, the protein / 3A4 treated for 72 hours at 37 ° C had been completely converted to the β-lamella conformation (see the dashed line in Figure 12).

• METHOD OF SELECTION, ETIOUETATION AND DETECTION OF THE ANTIBODIES THAT WERE USED IN THE TRIAL In other places, the protocols and methods for the production and characterization of antibodies are generally described [Harlow and Lane (1988) supra: 726]. The data described in this and the following examples were generated with the monoclonal antibody 6F3D, developed against the synthetic peptide corresponding to the sequence of the human A4 protein (Research Diagnostics Inc., Flanders, NJ). However, monoclonal antibodies made against the synthetic analogue of the ßA4 protein can also be used. Denatured forms of the ßA4 protein that recognize recombinant Fab can also be used. The protein / 3A4 in the a-helical conformations, of 3-lamella and random coil was attached to the polystyrene plates activated by glutaraldehyde, and incubated with the primary antibody diluted in series. The amount of IgG that reacted with each conformation of / 3A4, with anti-rabbit or anti-mouse antibody labeled with europium was determined, in accordance with the usual protocols, and the total signal was measured by resolved fluorescence by time, of enhanced dissociation . To develop the assay, antibodies were selected with the signal ratio of the denatured conformation against that of / S-lamella of ßA4 equal to or greater than 2.

COMPETITIVE AND DIRECT TRIAL FORMAT In the direct assay, each sample of the dilution curve of the ce-helical and / 3-lamella forms of the protein / 3A4 was divided into two aliquots: (1) untreated (designated native); (2) mixed with 4M Gdn * HCl / 1 percent final Sarcosyl, and heated for 5 minutes at 100 ° C (designated "denatured"). Both samples were diluted 20 times with H20, and the aliquots were loaded onto the polystyrene plate activated with 0.2 percent glutaraldehyde, for 2 hours.

The plates, incubated overnight at 5 ° C, were blocked with TBS, at a pH of 7.8, containing 0.5 percent BSA (weight / volume) and Sorbitol at 6 percent (weight / volume). The samples were then washed three times with TBS, at a pH of 7. 8, which contained 0.05 percent (volume / volume) of Tween® 20, and were incubated with the primary antibodies against the ßA4 protein (6F3D, Research Diagnostics Inc., Flanders, NJ). The samples were washed and then developed with secondary antibodies labeled with europium against mouse IgG (Wallac Inc., Turku, Finland). The plates were developed after about 7 additional washing steps in the breeding solution (Wallac Inc., Turku, Finland) and the signal was counted in the DELFIA 1234 Fluorometer (Wallac Inc., Turku, Finland). The results for the analytes indicate a marked difference in both the available binding sites and. in affinity for anti-3A4 IgG with different conformations of the? A4 protein. The method can be performed using a competitive assay, where the analyte / 3A4 is previously incubated, in different conformations, with anti-3A4 IgG, and then transferred to the polystyrene plate coated with synthetic ßA4 protein in the denatured state of Gdn - HCl.

DIFFERENTIAL TEST FOR DIFFERENT CONFORMATIONS OF < 3A4 The parameters obtained from the direct assay with the 6F3D anti-3A4 IgG were plotted as a function of the concentration. The results obtained with ¿SA4 in the 3-helical conformation (Figure 13) show a large difference between the protein signal in 3-lamella and denatured. The limit of sensitivity for denatured / 3A4 is = l μg / milliliter, and the range of linearity is about 2 orders of magnitude. In experiments with the ce-helical form of / 3A4 (Figure 14), 6F3D IgG binds equally strongly to both native and denatured forms of the protein. In contrast, the reactivity with the native / 3-lamella form of the protein only marginally exceeded the background up to high protein concentrations. When the results are expressed as a proportion of the fluorescence of the denatured states against native ßA4 (Figure 15), the proportion for ce-helical conformation is = l, and for / 3A4 in the conformation of ß-lamella is in a given concentration range of = l .5. This effect was also used to analyze samples of ßA4 of unknown conformation, where the small increase in the signal of 3F4 IgG labeled with europium, after the denaturation of ßA4, is a characteristic of the ce-helical conformation. In contrast, the large increase in the signal on the expected change for the ce-helical conformation is diagnostic of the 3-lamella conformation (Figure 15). The results can be expressed in two different ways: (1) as a ratio (Figure 15), where the index = l.O to / 3A4 indicates that the protein was originally in all the ce-helical conformation, and the index >; 1.5 indicates the presence of the / 3-lamella conformation; (2) according to the formula shown above, wherein the increase in excess of the signal over that expected for a ce-helical conformation is proportional to the amount of / 3A4 in the conformation of ß-lamella.

EXAMPLE 13 DIAGNOSIS OF ALZHEIMER DISEASE BASED ON INCREMENT OF THE PROTEIN TOTAL ßA4 ON PHYSIOLOGICAL LEVELS The exact quantitative measurement of total ßA4 by means of exclusively evaluating the signal of the denatured sample is apparently more accurate than the measurement of / 3A4 in the native conformation (Figures 13 and 14). The normal value of the protein can hide a significant portion of the β-plate form of the ßA4 protein due to the lower reactivity of this form with the antibodies. Because the only known condition for the accumulation of protein / 3A4 is the accumulation of the 3-lamella form of / 3A4 in the brains of patients with Alzheimer's disease, the increased levels of / 3A4 total in the sample proven is indicative of the presence of pathogenic forms of ßA4. This ßAA assay can be used in tissue, body fluids, or a pharmaceutical sample suspected of containing the / 3-lamella forms of / 3A4.

DIAGNOSIS OF ALZHEIMER'S DISEASE BASED ON THE INCREASE, OF THE SIGNAL PROPORTION BETWEEN ßA4 DESNATURALI ADA "AGAINST NATIVE (" AMILOID INDEX OF <3A4") The ratio between the affinity of the antibody for the ßA4 protein of denatured human against native is in the wide range of concentration for 6F3D IgG between 0.8-1 The proportion for ßA4 in / 3-lamella is through the range of complete linearity, over these values (Figure 15) .The "amyloid index of jß 4" gives a relative indicator of the presence of ßAA in 3-lamella insoluble pathogen.This assay mode of ßAA can be used directly in the brain, tissue samples, body fluids or pharmaceuticals, after the determination of the index for normal controls DIAGNOSIS OF ALZHEIMER'S DISEASE FROM THE DIFFERENTIAL ASSAY BY CALCULATING THE CONTENT OF ßAA By using the direct test and the formula shown above. Thus, the number of pathogenic forms of the ßAA protein in the foil in the brain, tissue samples, body fluids or pharmaceuticals can be calculated. EXAMPLE 14 MEASUREMENT OF THE CONVERSION OF A-HELICOIDAL TO B-LAMINILLA OF THE PROTEIN ßAA IN VITRO TO TRACE THE POTENTIAL DISEASE THERAPEUTICS The aliquots of a solution of 350 were incubated μg / milliliter of the ce-helical form of the synthetic ßAA (1-40), or the corresponding recombinant or synthetic peptides of the ßA protein, in PBS, with a pH of 7.4, for 72 hours at 37 ° C with concentrations of 10 ~ 3 - 10-6 M glycerol, cyclodextrins, heparin, heparin sulfate, Congo Red, cholesterol ester, dimyristoyl phosphatidylcholine. The samples were then divided into two aliquots: (1) untreated, containing 1 percent Sarcosyl and designated "native."; and (2) mixed with 4M Gdn HCl / 1 percent final Sarcosyl and heated for 5 minutes at 100 ° C, designated "denatured". Both samples were diluted 20 times with H20, and the aliquots were loaded onto the polystyrene plate activated with glutaraldehyde. The plates, incubated overnight at 5 ° C, were blocked with TBS, at a pH of 7.8, containing 0.5 percent BSA (weight / volume) and 6 percent Sorbitol (weight / volume). In the next step, these were washed three times with TBS, at a pH of 7.8, containing 0.05 percent (volume / volume) of Tween® 20, and incubated with the 6F3D IgG, and then with labeled anti-mouse antibody. with europium. The plates were developed after about 7 additional washing steps in the breeding solution, and the signal was counted in the DELFIA 1234 Fluorometer (Wallac Inc., Turku, Finland). The degree of conversion of the ce-helical conformation to that of / S-lamella of the ßAA was calculated from "amyloid index" (Figure 15), or alternatively from the formula shown above. Any compound that inhibits conversion by means of stabilizing the ce-helical conformation of the protein / SA4 may have therapeutic potential in vivo, by preventing the formation of mature amyloid.

EXAMPLE 15 EST NDARS OF NORMAL AND AMILOID TTR FORMS Soluble forms of TTR were obtained with Sigma Chemical Comp. A portion of the protein was solubilized in 100 mM KCl pH buffer, at a pH of 7.4, at the final protein concentration of 0.5 milligram / milliliter; this protein was designated "normal", and stored at -80 ° C until used. The second portion of the protein was converted to amyloid as described [Lai, Colon et al. (1996) Biochemistry 35 (20): 6470-82]. Briefly, the protein was resuspended in 100 mM KCl pH buffer, which contained 50 mM sodium acetate, at a pH of 4.4, at a protein concentration of 0.2 milligrams / milliliter, and incubated for 72 hours at 37 ° C. This portion of the protein was designated "amyloid" and stored until used at -80 ° C. The turbidimetry and Congo Red fixation test verified the efficient conversion to amyloid [Lai, Colon et al. (1996) Biochemistry 35 (20): 6470-82]. Both proteins were used as standards for the development of the trial, and to establish the sensitivity and range of linearity.

DIRECT TRIAL FORMAT In other places, protocols and methods for the production and characterization of antibodies are generally described. The data described in this and the following examples were generated with the commercially available polyclonal antibody, developed against purified human TTR (Accurate Chemical and Scientific Corporation, Westbury, NY). In the direct assay, each protein sample taken from the dilution curve of the normal and amyloid forms of TTR was divided into two aliquots: (1) untreated, and designated "native"; and (2) mixed with 4M Gdn HCl / 1 percent final Sarcosyl and heated for 5 minutes at 100 ° C, and designated "denatured". Both samples were diluted 20 times with H20, and the aliquots were loaded onto the polystyrene plate activated with 0.2 percent glutaraldehyde for 2 hours. The plates, incubated overnight at 5 ° C, were blocked with TBS, at a pH of 7.8, containing 0.5 percent BSA (weight / volume) and 6 percent Sorbitol (weight / volume). In the next step, these were washed three times with TBS, at a pH of 7.8, which contained 0.05 percent (volume / volume) of Tween® 20, and were incubated with primary antibodies against TTR (Accurate Chemical and Scientific Corporation, Westbury, NY), were washed and then developed with secondary antibodies labeled with europium against rabbit IgG (Wallac Inc., Turku, Finland). The plates were developed after about 7 additional washing steps in the breeding solution, and the signal was counted in the DELFIA 1234 Fluorometer (Wallac Inc., Turku, Finland).

EXAMPLE 16 DIAGNOSIS OF SSA AND FAP BASED ON INCREASE OF THE PROPORTION OF SIGNAL BETWEEN TTR DESNATURALI ADA AND NATIVE ("TRO AMILOIDEO INDEX") The ratio between the affinity of the antibody for TTR of denatured human versus native is in the wide range of concentration for the polyclonal antibody of 1-3: 3. The ratio for the amyloid form of TTR is through the range of complete linearity between 0.7-1.0 (Figure 18). The "TTR amyloid index" gives a relative indicator of the presence of pathogenic TTR, insoluble and forming amyloid. This TTR assay mode is being used directly in the brain, tissue samples, body fluids or pharmaceuticals, after index determination for normal controls.

DIAGNOSIS OF SSA AND FAP FROM THE DIFFERENTIAL TEST THROUGH THE CALCULATION OF TTR AMYLOIDEA CONTENT By using the direct test formula (Figure 19), the amount of the amyloid form of TTR can be calculated in the peripheral nerves, samples from tissue, body fluids or pharmaceutical products. In the formula (Figure 19), the signal increase less than expected for normal conformation is proportional to the amount of TTR in the amyloid conformation.

EXAMPLE 17 MEASUREMENT OF TTR IN VITRO NORMAL-A-AMYLIDE CONVERSION TO TRACE THERAPEUTICS POTENTIAL DISEASE The aliquots of a 0.2 milligram / milliliter solution of the normal form of synthetic TTR, or the corresponding recombinant or synthetic peptides of the TTR, were incubated in 100 mM of KCl pH buffer, containing 50 mM of acetate of sodium, with a pH of 4.4, for 72 hours at 37 ° C with concentrations of 10 -3 10 -6 M of organic compounds tested. The samples were then divided into two aliquots: (1) untreated, containing 1 percent Sarcosyl and designated "native"; (2) mixed with 4M Gdn HCl / 1% final Sarcosyl and heated for 5 minutes at 100 ° C, designated "denatured". Both samples were diluted 20 times with H20, and the aliquots were loaded onto the polystyrene plate activated with glutaraldehyde. The plates, incubated overnight at 5 ° C, were blocked with TBS, at a pH of 7.8, containing 0.5 percent BSA (weight / volume) and 6 percent Sorbitol (weight / volume). In the next step, these were washed three times with TBS, at a pH of 7.8, containing 0.05 percent (volume / volume) of Tween® 20, and incubated with the polyclonal anti-TTR antibody (Accurate Chemical and Scientific Corporation , Westbury, NY), and then with anti-rabbit antibody labeled with europium. The plates were developed after about 7 additional washing steps in the breeding solution, and the signal was counted in the DELFIA 1234 Fluorometer (Wallac Inc., Turku, Finland). The degree of conversion of the normal to amyloid conformation of the TTR was calculated from the "amyloid index" (Figure 18), or alternatively from the formula (Figure -19). Any compound that inhibits conversion by stabilizing the normal conformation of TTR may have therapeutic potential in vivo, by preventing the formation of mature amyloid.

EXAMPLE 18 TYPIFICATION OF PRISON ISOLATES (CEPAS) IN SYRIAN HAMPSTERS Syrian hamsters (LVG / LAK) were infected by intracerebral injection of the following isolates from scrap adapted to the hamster, that is, different groups of hamsters were infected with individual strains of prions as follows: Drowsy (Dy), 139H, Hyper (Hy), Me7, MT-C5, and Sc237. Animals were euthanized in the final stages of the disease and their brains were immediately frozen and stored at -70 ° C. The brains were homogenized on ice in 3x30 seconds touches of the PowerGen homogenizer (Fisher Scientific, Pittsburgh, PA) in PBS, at a pH of 7.4, containing a cocktail of protease inhibitors (5mM PMSF, Aprotinin and Leupeptin 4 μg / milliliter). ). The resultant 10 percent (weight / volume) homogenates were rotated for 5 minutes at 50 ° C in the tabletop centrifuge. The supernatant was mixed at 1: 1 with 4 percent Sarcosyl in PBS, with a pH of 7.4, and divided into two aliquots: (1) untreated and designated native; (2) mixed with a final concentration of 4M Gdn HCl and heated for 5 minutes at 80-100 ° C and designated denatured. Both samples were diluted 20 times with H20, and the aliquots were loaded onto the activated polystyrene plate for 1 hour with 0.2 percent glutaraldehyde in PBS. The plates, incubated overnight at 5 ° C, were blocked with TBS, at a pH of 7.8, containing BSA_ at 0.5 percent (weight / volume) and Sorbitol at 6 percent (weight / volume). In the next step, these were washed three times with TBS, at a pH of 7.8, containing 0.05 percent (volume / volume) of Tween® 20, and incubated for 2 hours with the monoclonal 3F4 antibody labeled with europium. The plates were developed after about 7 additional washing steps in the breeding solution provided by the Europium label supplier (Wallac Inc., Turku, Finland), and the signal was counted in the DELFIA 1234 Fluorometer (Wallac Inc., Turku, Finland). The content of prpSc and e - [_ "prion index" (ratio of the binding of the antibody to the denatured PrP protein: native) was calculated as described in Examples 11 and 8, and plotted on the xy coordinates as shown in FIG. shown in Figure 15. It can be assumed that other samples tested and that resulted in the same calculations have the same prion strain in them. The present invention is shown and described herein in which they are considered to be the most practical and preferred modalities. It is recognized, however, that deviations from them can be made, which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art after reading this description.

Claims (10)

1. CLAIMS 1. A method for determining the presence of a pathogenic form of a selected protein, in a sample comprising a first non-pathogenic conformation of the protein and a second pathogenic conformation of the protein, the method comprising: contacting a first portion of the sample with a binding partner having a greater affinity for the first conformation than for the second conformation, and determining a first concentration; treating a second portion of the sample to increase the binding affinity of the second conformation to the binding partner; contacting the second treated portion of the sample with the binding partner to determine a second concentration; adjusting the second concentration to provide an adjusted concentration, adjustment compensating for the increased affinity of the protein in the first conformation for the binding partner resulting from the treatment; and comparing the first concentration with the adjusted concentration, to determine the presence of the protein in the second pathogenic conformation.
2. 2. The method of claim 1, wherein the sample is obtained from an animal that does not exhibit symptoms of » 109 disease; wherein the first concentration and the second concentration are determined using time resolved, enhanced dissociation fluorescence; 5 also wherein the second pathogenic conformation of the protein is present in the sample at a concentration of 1 x 103 particles / milliliter or less; also wherein the protein is selected from the group consisting of the ßAA protein, the PrP protein, and transthyretin.
3. The method of claim 1, wherein the protein is fixed to a solid surface, and wherein the treatment comprises subjecting said sample to a treatment selected from the group consisting of heat, pressure, fifteen . and chemical denaturation, sufficient to convert at least 2 percent of any protein in the second form to the binding form; wherein the binding partner comprises a labeled antibody that has an affinity for the first form when 20 times ten times greater than their affinity for the second form.
4. 4. A method for determining the presence of a pathogenic form of a selected protein, in the presence of the protein in a non-pathogenic conformation, wherein the protein exists in a first conformation and in a second 25 conformation that differs in binding affinity to a binding partner, the method comprising: treating the sample to convert the second conformation of the protein to a binding conformation having an affinity for a binding partner higher than the second conformation; contacting the treated sample with the binding partner to determine a concentration; adjusting the concentration to provide an adjusted concentration that compensates for the increased affinity of the first conformation of the protein to the binding partner that is the result of the treatment; and comparing the first concentration adjusted to a known concentration selected from the group consisting of a control concentration and a previously determined standard concentration, to determine the presence of the protein in the second conformation in the sample.
5. The method of claim 4, wherein the binding partner comprises a labeled antibody, and wherein the concentration is determined using flow cytometry; wherein the adjusted concentration is compared to a known known concentration of a treated uninfected control sample, or compared to a previously determined known concentration of a treated sample from an uninfected population; also wherein the antibody is 3F4; still further where the protein in the second conformation is present in the sample at a concentration of 1 x 103 protein molecules or less per milliliter, and wherein the protein in the first conformation is present in the sample at a concentration of 1 x 106 or more protein molecules per milliliter.
6. 6. A method for identifying a compound having a therapeutic activity against a prion-mediated disease, the prion-mediated disease characterized by a protein having a first form and a second form differing in conformation, one of said forms-being associated with prion-mediated disease, the method comprising: providing an animal susceptible to a prion-mediated disease; administering a test compound to said animal; induce prion-mediated disease; get a sample of that animal; contacting a first portion of a sample containing the protein with a binding partner, the binding partner having a greater affinity for that first form of the protein than the second form, and determining a first concentration; treat a second portion of the sample to convert the second form to a fixation form that has greater affinity for the fixation partner; contacting the second treated portion with the binding partner to determine a second concentration; adjusting the second concentration to provide an adjusted concentration that compensates for the increased affinity of the protein in the first form for the binding partner that is the result of the treatment; and comparing the first concentration with the adjusted concentration to determine the conformation of the protein in the second form, and deducting the influence of the test compound on the concentration of the protein in the second form.
7. 7. A method for determining a strain of a pathogenic protein in a sample, comprising: determining the concentration of a pathogenic conformation of a protein in a sample; treating the sample in a manner for the purpose of changing the pathogenic conformation of the protein to a conformation having a higher binding affinity for a binding partner than the pathogenic conformation; determine the influence of the treatment on the protein in the pathogenic conformation; and compare the determined influence with the known standard influence for a known strain at a known concentration, and by the same deduce the strain from the Pathogenic conformation of the protein in the sample.
8. 8. A compound identified by the method of any of claims 6 and 7, characterized by the reduction of the protein concentration in the second form.
9. 9. A method for determining a strain of a pathogenic protein in a sample, comprising: determining the concentration of a pathogenic conformation of a protein in a sample; treating the sample in a manner for the purpose of changing the pathogenic conformation of the protein to a conformation having a higher binding affinity for a binding partner than the pathogenic conformation; determine the influence of the treatment on the protein in the pathogenic conformation; and comparing the determined influence with a known standard influence for a known strain at a known concentration, and thereby deducing the strain from the pathogenic conformation of the protein in the sample. The method of claim 9, wherein the concentration of the pathogenic conformation and the influence by treatment are determined using an antibody labeled as the binding partner, and using time resolved, enhanced dissociation fluorescence; wherein the pathogenic conformation of the protein is PrPSc and the PrPSc is treated with proteinase K.