UA76115C2 - Early diagnostics of conformational diseases - Google Patents

Abstract

A method for the diagnosis or detection of conformational diseases by assaying for a marker (the pathogenic conformer) of such diseases in a sample is described, which method comprises a cyclic amplification system to increase the levels of the pathogenic conformer which causes such diseases. In particular, such transmissible conformational diseases may be prion encephalopathies. Assays, diagnostic kits and apparatus based on such methods are also disclosed.

Description

The tumor underlying the disease is a prion The tumor underlying the disease is the prion protein. white. 13. A method of detecting the presence of a pathogenic form 22. A method of diagnosing Alzheimer’s disease in a prion protein in a sample, which includes: a patient, which includes taking a sample from a patient - (Y) contacting the specified sample with a certain amount of: non-pathogenic prion squirrel; (I) contacting the sample with a certain amount of non-patho- (ia) incubation of the sample/non-pathogenic prion gene B-amyloid protein; squirrel; (ia) incubation of the sample/non-pathogenic B- (i) disaggregation of any amyloid protein-forming aggregates; occur during stage (ia); (i) disaggregation of any aggregates forming - repetition of stages (ia)-(i) two or more times; and they occur during stage (ia); then repeating stages (ia)-(ii) two or more times; and (ii) determining the presence and/or amount of pathogenic prion protein in the sample. (iii) determination of the presence and/or amount in the sample 14. The method according to item 13, where the cycle that is formed is a pathogenic β-amyloid protein. by actions (ia) and (ii), is repeated from 5 to 40 times up to 23. The method according to item 22, where the cycle that is formed is the execution stage (its). actions (ia) and (ii), is repeated from 5 to 40 times up to 15. The method according to item 13, where the pathogenic conformer is the performance of stage (ii).

Re, the non-pathogenic conformer is RgRe, and white 24. The method according to paragraphs 22, 23, where the pathogenic conformer underlying the disease is the prion rum is RgRze, the non-pathogenic conformer is RgRU, and the protein. the protein underlying the disease is the prion protein.

Jacob's disease in a patient, which includes taking a sample 25. The method of analysis to detect a marker of comfort in a patient: mation disease, which is characterized by (i) contact of the sample with a certain amount of protein by conformational transformation of the lying protein

Rg?Re; at its core, from a non-pathogenic to a pathogenic con- (ia) incubation of the sample/RhRe protein; former in the sample, where the specified analysis includes (i) disaggregation of any aggregates that form the following stages: during stage (ia); () contacting the specified sample with a certain number of repetitions of stages (ia)-(i) two or more times; and a non-pathogenic conformer; then (i) disaggregation of any aggregates that form - (i) determination of the presence and/or quantity of PgRzs used in the final result during stage (i); and sample. (ii) determination of the presence and/or quantity in the sample 17. The method according to item 16, where the cycle formed by the specified pathogenic conformer, and by patho-actions (ia) and (ii), is repeated from 5 to 40 times until the genetic the conformer is a marker of the presence of the specified stage (its) execution. 18. The method according to item 16, where the pathogenic conformer is stage (ia) of incubation of the specified

PgrPzs, the non-pathogenic conformer is PgR, and the protein of the sample/non-pathogenic conformer, and stage (ia) and the tumor underlying the disease is prion (ii) form a cycle that repeats every protein. nshe twice before performing the stage (her). 19. The method of detecting the presence of a pathogenic form 26. The method of analysis according to item 25, where the cycle that forms

of B-amyloid protein in the sample, which includes: stages (ia) and (ii), repeated from 5 to 40 (І) contact of the sample with a certain number of nepato times before performing stage (ii). gene B-amyloid protein; 27. The method of analysis according to any of paragraphs 25, 26, where (ia) incubation of the sample/non-pathogenic B- pathogenic conformer is PgRes, a non-pathogenic amyloid protein; the conformer is PgR-, and the protein underlying the disaggregation of any disease-forming aggregates is the prion protein. occur during stage (ia); 28. Diagnostic kit for use in the analysis of repetition of stages (ia)-(ii) two or more times; and according to any of paragraphs 25-27, which includes a certain amount of the non-pathogenic conformer, multi-well determination of the presence and/or amount in the sample microtiter plate and multi-well pathogenic B-amyloid protein. ultrasonic device. 20. The method according to item 19, where the cycle formed by sta- 29. Apparatus for use in the diagnosis of con- diseases (ia) and (ii) is repeated from 5 to 40 times until firmation disease according to any of items. 1- performance of the stage (its). 27, which includes a microtiter tablet, 21. The method according to item 19, where the pathogenic conformer is a multi-well ultrasonic device and an excess

PgrPzs, the non-pathogenic conformer is PgR, and the protein amount of the non-pathogenic conformer.

The present invention relates to a method for diagnosing and increasing the levels of a pathogenic conformer. Zotics or the detection of conformational diseases of Rhema, the specified conformational diseases can be done by analyzing the sample for the presence of a marker (ie, they represent prion encephalopathies. that is, a pathogenic conformer) of the specified diseases. Conformational diseases are vans; the specified method includes the use of a group of disorders that do not have an obvious connection of the cyclic amplification system to each other, but have a pronounced similarity in clinical manifestations, which reflects their common requests, complicated by the apparent absence of immune mechanisms of stimulation and self-association, with no response to RgRes, Clinical diagnosis of UTI on subsequent deposition and tissue damage. today is based on a combination of subhost-

Interest in the structure arises due to the swarm of progressive dementia (in less than 2 years), the fact that the indicated various diseases cause myoclonus and multifocal neurological dysa by the conformational transformation of the protein, which functions associated with the characteristic periodic underlies them, which usually leads to its electroencephalogram (EEG) (WHO report, aggregation and deposition in tissues. From medical 1998, Uureg eyi a!I., 1997). However, the version of the SOO point of view, the manifestation of the specified conformational (mSO0), most iatrogenic forms of SOI and up to 4095 diseases reflects this molecular sporadic cases do not show EEG abnormalities mechanism, usually with a slow onset, which is non-Ieieipnoiy ei ai., 1996). On average, the accuracy of clinical-significantly occurs when the conversion of observational diagnosis is about 6095 for SOJ and is in a normal protein, but with a more sudden high variable for other associated with the onset, when it is observed in unstable prion diseases. Clinical diagnosis is a protein variant. Transmissible spongiform encephalopathies and deme- 1997 are two examples of special, more precise only in the later stages of the disease- importance of the specified conformational diseases, when clear symptoms develop (UMebeg ei ai., 1997). Alzheimer's disease is a disease that threatens Genetic analysis is suitable for diagnos- become devastating for health care systems in ki hereditary prion diseases, but the latter developed countries (see review Saitei! ei a!., 19971. represent only 1595 of all cases Methods

Transmissible spongiform encephalopathies of CNS imaging are suitable only for exclusion (T5E), also known as prion diseases, manifestations of other states of rapidly progressive dementia, are a group of neurodegenerative diseases due to structural damage to the brain that affect humans and animals. Brain disease (Muereg ei ai., 1997). Findings made by

Creutzfeldt-Jakob (CO), kuru, Herst's disease - using techniques that visualize the main Mann-Straussler-Shaker (555) and fatal brain, by computed tomography (CT) and familial insomnia (ER) in humans, and also 5sgarie todyks of nuclear magnetic resonance (NMR), za- (pruritus) and spongiform encephalopathy lie, mainly, from the stage of the disease. cattle (B5E) in animals are a de- CT is a much less sensitive method, and in the early which of T5E diseases (Rgizypeg, 1991). stage in 8095 cases, atrophy is not detected

In spite of the fact that these diseases are rare, ISaime2 apa Sapieg, 1983). In addition to atrophy, the findings are relatively rare, the risk of possible superintense NMR signals on the basal ganglia of human VE along the food chain is taken up by the ganglia (Opotgii et al., 19931. As well as the changes observed by the health authorities I and are treated with the help of CT, these changes are not specific in any way by any scientific community.

Vgyse eyi a!., 1997). Recent research has provided an opportunity

These diseases differ too tri- to identify several proteins of neurons, astrocytes with a general incubation period, followed by cor- and glia, the content of which increases with SOJ |itis ei ai., tka and inevitably fatal clinical stage of the disease 19921. Protein levels 5-100, neuron-specific iso- (Koo5 ei ai., 1973). Today, enzyme and ubiquitin treatments are significantly increasing in this- not developed. ribospinal fluid (C5E) in the early stage of

The key characteristic of this disease is the formation of a protein of a pathological configuration, with increasing concentrations in the course of the disease (Iti et al., 1992). A death marker called Ргрз-, which is a post-transneuron protein 14-3-3, has been proposed as a splicing modified version of the normal protein, a specific and sensitive test for sporadic SOY called РгРУ |Sopep apa Rgizipeg, 19981. INzisp ei ai ., 19961. However, it is not suitable for dia-

For distinguishing isoforms of PIR, no gnostics of MSO)O were found and it is much less specific for chemical differences (Ziapyi ei Ai., 1993) and convergent genetic forms. Since the 14-3-3 protein can be present in C5E patients with other conditions, it appears to be a conformational change, while the a-helical content of nor- this test is not recommended by the WHO as the total small protein decreases, and the number of p-screening for SO and remains in reserve for subt-folded structure is increasing |Rap eyi ai., the development of clinical diagnosis (WHO report, 1993|Y. Structural changes are followed by changes in 1998). biochemical properties: RgRO is soluble in de- More successful diagnosis is achieved with non-denaturing dotergents, RgRO is insoluble; using a combination of clinical and biochemical data

PgR is easily cleaved by proteases, while markers. However, according to the operative diagnosis

RgRes is partially resistant, which leads to the nostics accepted today in Europe - the formation of an M-terminally truncated fragment, the Peian system of observation of SOO, the latter known as the "RgRgev" form | Vaidu/pei ai., 1995; The diagnosis is established only by

Sopep apa Rgizypeg, 1998), "Rgr 27-30" (27-ZOkDa) according to the results of neuropathological studies and is or "RC-resistant" (resistant to protein - detection of RgRUs using immunohistochemical tests). techniques, histoblotting or western blotting

To date, accurate diagnosis of T5E (Mereg e! a!., 1997, ViakKa e! a!., 1995). there is no WHO report, 1998, VyakKa ei ai., The formation of RgRZs is not only the most 1995, Mereg ei ai., 1997). Attempts to develop a diag- more likely cause of the disease, but also a nostic test to detect prion diseases- the most well-known marker. The detection of Rg in tissues and cells is widely correlated with the pathological transformation of the protein underlying it and with the presence of TE infectivity, and methods of treatment that inactivate or eliminate the infectious agent by analyzing the sample for the presence the presence of the marker T5E, also eliminate RgR: |Rgivipeg, 19911. of the indicated disease; the specified method includes

Identification of RgR:: in the tissues of a person or an animal in itself: we are considered key for the diagnosis of T5E (before () contact of the specified sample with a certain WHO report, 1998. I1. An important limitation of the distance of the non-pathogenic conformer; this approach is sensitivity, because of the amount of RgRZ: there is (its) disaggregation of any aggregates that are alarming (sufficient to detect the usual ones in the final result during stage (1), and methods) only in the CNS in the late stages of the disease determination of the presence and/or amount in the ripening. However, it was shown that in the early stage of the specified pathogenic conformer, the actions of the disease take place generalized. of the specified disease. to the lymphoreticular system (Adigli, 1997). Indeed, Mostly stage () includes the stage reported on the presence of PgR:: in the tissue under- (a) incubation of the specified sample/non-pathogenic palatine tonsils and appendices obtained from a compliant patient. ntiv from MUSUO Ts|NIII ei ai., 1997). Despite the fact that according to the preferred embodiment, it is not known how early in the course of the disease of the modern invention, stages (ia) and (i) form a cycle, it is necessary to use a biopsy of the tonsils or appendix, which is repeated at least twice before performing the diagnosis mSOYU, it was shown that it is in stage (iii). More preferably, the cycles of sheep genetically predisposed to the disease repeat from 5 to 40 times, and most preferably - 5- burns (itching), RgRZe can be detected in the tonsils - 20 times. lar tissue even before the appearance of symptoms and in the early stages of conformational diseases subject to the incubation period. However, until now detection or diagnosis, are the following

RgRZs was not detected in the indicated tissues in women with diseases that are characterized by a conformational case of sporadic ACS or by social transformation of the protein underlying them

IKamzhavznita eyi a!., 1997). you This "protein underlying" is

The normal protein is expressed by white blood cells, a protein that is able to perceive non-pathogenic cells and platelets, and, therefore, possible inflammation and pathogenic transformation. An example of some cage-like protein in individuals affected by the disease is the prion protein, PgR. Another type of blood can contain RgRe: Iadii, 1997). This is an example of a similar protein is a protein that has the possibility of developing a blood test for CO, bearing on Alzheimer's disease, that is, VD- but requires research with a much higher amyloid protein. higher sensitivity than the sensitivity of research methods that exist today. diagnosis or detection, represent a re-

It is assumed that the replication of prions observed in transmissible conformational diseases occurs when RgR:: in the infecting inoculum, spe- such as TZE (as defined in the "Prerequisites" section). in a specific way interacts with the PgR of the host, catalyzes in the case of diagnosis of T5E and in accordance with its transformation into the pathogenic form of the protein of the preferred version of the embodiment of this invention, (Sopep eyi a!., 1994). In order to achieve the concentration of the marker of this disease, as well as the pathogenesis of RgRZs, sufficient for the appearance of clinical symptoms, the conformer is RgR:-, while non-pathogenic, the specified process continues during this conformer of the protein of interest , for a period of time from many months to many years. kiv The amount of non-pathogenic conformer, which

The infectious unit of RgR:- is believed to represent used in stage (i) (and, optionally, an oligomeric structure with a high content of B- stage (iB)), this is usually a known amount, although it is a folded structure that converts the normal not necessarily in the case that it is necessary to simply establish by including it in the growing aggrenovate the presence or absence of a pathogenic congate (Fig. 1). This conversion is simulated by a do-former. by mixing purified PgR- with a 50-fold It is preferable that the amount of non-pathogenic former denatured by a conmolar excess in advance, which is used in stage (ii) (i, non-

Rgrz: (Kosivko eyi a!., 1994). necessarily, at stage (IB)), there was an excessive amount of

The IP conversion systems described so far. In general, the initial ratio of mygo have low efficiency, since they require an excess of PgrR: and therefore are not suitable for the mayor (if it is present in the sample) will be more diagnostic purposes, since they cannot determine 1001, mainly , more than 1000:1, and the most detectable amounts of the marker. Importantly, more than 1,000,000:1. The reason for the low efficiency is that the number of oli- In another preferred embodiment of the RiRUs homers (converting units) of this invention, the non-pathogenic conformer remains fixed during the study. Odi- action () is present in the brain homogenate of the converting cells, which grow sequentially on the kinary subject and/or can be added to it, and as a result, they become larger, but the keel- before the implementation of the stage (th); in this case, their thickness does not increase (Fig. 1). same, the homogenate of the brain, which contains (pe-

The applicants have developed a method of diagnosis or, rather, known) excess of a non-pathogenic conformational disease, when the former is added at stage (ii). Preferably, which disease is characterized by a conformation, the homogenate of the brain of a healthy subject has the same species of origin as that of the sample that analy- a!., 19971. After incubation of two forms of PgR, oligo-human for a human sample, homogenate, large-sized species increase their size due to a rat brain for a rat sample, which is subject to radiation and transformation of PgR molecules. This analysis) It is more preferable that the non-pathogenic process has a low efficiency, because it conformer was present in a specific fraction lies from a fixed number of oligomers that homogenate the brain, for example, in fatty tissue at the ends. The number of converting units, masses with of brain homogenate. The yield increases during the reaction during which they only given fractions can be carried out, for example, as they become larger in size. It is assumed that yes- (described in Zagdia soto M. ei a!., 19931. ny process is what happens in

Thus, this invention also applies to the body of an animal or a person after infection; to the method or analysis as described in this do- a process that is known to take months or even document in which the tissue or tissue fraction several years. In this invention, the applicants describe adding to the non-pathogenic conformer at the second stage the procedure of breaking down the oligomers into more (Y). It is preferable that the tissue is a tissue of small molecules, each of which is then able to convert the brain or the homogenate obtained from it to Pgr-. or a fraction from a healthy subject (that is, from such a subject who does not have a pathogenic confor- is used for the diagnosis of conformational mer). diseases, and, in particular, transmissible confor-

It is reported |Kosizko eyi a!., 1994| that less mation diseases, such as T5E, through the amglycosylated forms of RgRU mainly convert the amounts of Rgr:e in various tissues or into the form of RgRzs, In particular, RgRsO, to which biological liquids, which are not detected by other methods of specific phosphatidylinositol. This system can allow early phospholipase C, of course, more effective detection of people who are at risk of transformed into a pathogenic form, than full, development of TOE, and could also be quite useful- more intensively glycosylated, PgR-. Thus, for the biochemical control of the effectiveness of noms, another variant of the implementation of this invention of drugs for the treatment of T5E during clinical refers to the method or analysis as described in these tests. according to this document, in which non-pathogenic con- According to the preferred embodiment, the former is PgR, which has a reduced level of glyco- given invention, the sample to be analyzed, prosylation (in particular, M-linked glycosylation) goes through the stage of "pretreatment ", the purpose of which is to compare with wild-type RgRO. Preferably, the RgRU "selective concentration" in the pathogen sample is processed to remove some, all or a significant conformer to be detected. In the case of the amount of glycosylation before its use by T5E, as reported, both PgRU and Pg5e are located as a non-pathogenic conformer in the methods and are analyzed in a special area of the plasma membrane, as described in this document; and, more resistant to treatment with mild detergents, the non-pathogenic conformer is a tame (such as the ice triton X-100), thanks to

RgRU, which is practically non-glycosylated. relatively high content of cholesterol and glycosyphil

In the case of diagnosis of TZE, if in the sample of lipids (M. Meu ei ai., 1996). The specified membrane essential aggregates of the pathogenic form, during stage (iii) the domains are called fat masses or stable, they will induce the conversion of PgPo-»RugRZe, and to detergents membranes (OKM) or caveolae - during stage (ii) the specified aggregates will be decay-like domains (SI-O), and they have a high tsity into smaller but infectious units, the content of signaling proteins, receptors, and CRI-anchors, each of which is still capable of inducing protein conversion. The applicants confirmed that 10095 RgR: in another RgRO. This technique in this document captures the brain in this fraction, which is called "cyclic amplification" and represents "290 of all proteins (see example 6 and Fig. 7). It is shown in Fig. 2. This system provides an exponential, simple stage of allocation of fat masses with a significant increase in the amount of PgR:s, in the final re- sample, it allows to realize a pronounced enrichment of the result present in the sample, which can be easily contained in PgRO. Similar results were obtained to detect. According to another preferred by the applicant regarding the allocation of fat masses from the variant of the embodiment of this invention, it is possible, brain mogenate during burning, from which was therefore, the calculation of the amount of RgRs, which is initially in the obtained Rgrzs, sample, based on the known amount of RgRU , determine- Thus, one version of the implementation of this given amount of Rgrzye, which is in the sample until the end of the analysis, the invention includes a stage during which immediately and taking into account the number of performed cycles. the object to be analyzed is in the stage of

On the contrary, if there is no Pgr: in the sample (as such a rare treatment for selective concentration in or in the form of aggregates), the RgR:U molecule does not transform into a pathogenic conformer. It is preferable to dig into PgRe, and by the end of the analysis, the marker will be from the pathogenic conformer was PgRje, the previous essence (pathogenic treatment will not be detected in the sample, the treatment represents extraction from the sample of fraconformer). cation, which is insoluble in mild detergents.

It was shown that the infectious unit Stages (ii) and (a) are mainly performed in physiological

RiRZs is an oligomer with a high content of the D-structure, under harsh conditions (pH, temperature and concentration, which can be transformed into a normal protein ion) and, more preferably, to the solution also by integrating it into the growing aggregate, where protease inhibitors and detergents were added. conditions, it acquires the properties inherent in the pathological form (resistance to proteases and insolubility) to any pathogenic conformer, if it is present in the sample, to convert the non-pathogenic conformer into the specified ultrasonic devices can be easily adapted to the pathogenic conformer, thus forming an aggregate or oligomer of pathogenic conformers for use in a diagnostic method according to the invention.

The appropriate physiological conditions can be easily determined by a specialist. of the invention refers to the application at stage (iii)

The duration of incubation in time will be such that the multiwell ultrasonic device. to allow a certain amount, the total amount or Detection of a newly converted pathogenic significant part of the non-pathogenic conformer of the over-conformer, for example RgRZe, (iii) after the procedure to turn into the pathogenic conformer, if the assumption of cyclic amplification described in stages (i1)-(ii), that the sample contains a certain amount of pathogenic can be carried out according to any of the vi- conformer. It is easy for a specialist to determine the specified home methods. Specific detection of RgRe: usual. Preferably, each incubation consists of teano (but not always, see below) takes place for 1 minute to 4 hours, most preferably - from 30 the first stage of separation of the two RgR isoforms (nor- minutes to 1 hour, and, especially preferably - approx. - small protein and pathogenic protein). Separation lasts 60 minutes. carried out on the basis of peculiar biochemical properties

Incubation stage (a) can also include in the characteristics of PgRs, which distinguish it from the majority, an additional stage (IB), which includes normal proteins of the body, namely: Pgr5: is often the addition of an additional amount of non-pathogenic protein resistant to processing by proteases, and he is not a nonconformist. occurs even in the presence of detergents, which are not

To disaggregate the aggregates during stage (ii) they are denatured. Therefore, the first stage after the process according to the invention can be used for amplification is usually removal or different methods. They include the pouring of PgR: from the sample - with the help of transparent treatment: treatment with solvents (such as dodecytase or with the help of centrifugation - for sodium sulfate, dimethyl sulfoxide, separation of soluble protein (PgRU) from insoluble - acetonitrile, guanidine , urea, trifluoroethanol, go (RgRzs). After this detection of RgR?- it is possible to carry out diluted trifluoroacetic acid, diluted with any of the following formic acid, etc.), modification of physical methods: chemical properties of the solution, such as pH, tem- A) Immunoblotting after 505-RACE. This is due to temperature, ion concentration, dielectric constant using a conventional procedure, well on, and physical methods, such as sonication, known to those skilled in the art, and using some of the ba- laser irradiation, freezing/thawing, many commercially available anti -RgR-antibody. French press, autoclaving, high pressure, B) EGIZA analysis. Solid-phase detection of mixing, soft homogenization, other types can be carried out with the help of simple ana-irradiation, etc. In accordance with this invention, lysis in which the samples are placed on a plate, followed by sonication is the preferred method. which reveal the number of RgRZs with use

Disaggregation can be carried out during the time of anti-RgR antibodies or, more preferably, during the time during which disaggregation of some of the EGISA sandwiches occurs, in which the initial amount, the entire amount, or a significant part of the aggrecan is covered with anti-RgR - by an antibody that is specific for the cells formed during stage (ii). There is no way to capture PgR from the sample, which is then necessary to disaggregate all aggregates at any stage of disaggregation using the second anti-PgR. With this method, the cell is an antibody. Both forms of EGISA can also be treated with labeled (radioactivity, fluorescein, biotin, etc.) anti-PgR antibodies for

The time of disaggregation can be easily determined by a specialist, and further increase in detection sensitivity. it may depend on the method of disaggregation, which C) Radioactive studies. Normal is used. Preferably, the disaggregation of the effective RgRU, which is used as a substrate for newts during the time period from 1 second to 60 minutes of the amplification procedure, can be labeled with radioactive isotopes (ZN, 14C, 355, 1251 and etc.) before the minutes, and, especially preferably - from 5 seconds to 10 after the start of the procedure, and after removing non-conver- minutes. If the disaggregation is carried out with the help of RgRU, it can be quantitatively determined by the radioactivity of ultrasonic treatment, then it prolongs the life of the newly converted RgRes. This procedure is preferably from 5 seconds to 5 minutes, and most often it is more quantitative and is not calculated for preferably - from 5 to 30 seconds. use of antibodies.

Ultrasound treatment was used earlier O) Fluorescent analyses. Normal as part of some methods of purification of PgR for the purpose of PgRU, which is used as a substrate for the process of increasing the solubility of large aggregates, but its amplification dures, can be marked with fluorescence - has never been described as a method of amplification with conversion probes before the start of the procedure, and after the PGR seed ip miigo removal of unconverted PgR-U can be quantified

The use of traditional ultrasound to determine the fluorescence of a newly converted device with a single probe faces the problem of RitRje. It is possible that the fluorescence analysis can process a large number of samples at the same time, how not to require the removal of unconverted PgR, which is required by the diagnostic test. Because the fluorescent properties of PgRO and Pgrze, ultrasonic devices with a 96-well format can be different due to the distinct conformation of microtiter tablets that provide ulmation of the two specified isoforms are on sale. transsonic treatment of all pits at the same time and E) Aggregation analyses. It is well known that they can be programmed for automatic operation. Reese (but not PgrRU) can aggregate to form amyloid fibrils or rod-like structures. (iii) determination of the presence and/or quantity in the form of

Therefore, the detection of RgRo: could be carried out according to the specified pathogenic conformer. In general, with the help of the methods used for the pathogenic conformer will be a marker for the quantitative determination of the formation of these types of ag- the presence of the specified disease. regattas, including electron microscopy, ensure that stage () should include staining with specific dyes (Congo cher- stage (ia), during which incubation with it, thioflavin 5 and t and tp) and the indicated sample/nonpathogenic conformer takes place. nephelometric analyses. According to the preferred embodiment

Aggregation assays do not require the stage of the distributed invention, stages (ia) and (i) form a cycle of two isoforms, since normal PgR, which is repeated at least twice before completion, aggregates. nth stage(s). More preferably, if the cycles repeat

F) Structural analyses. The most weighted are from 5 to 40 times, and most preferably - the difference between normal and pathogenic RgR 5-20 times. there are their secondary and tertiary structures. Therefore, another purpose of this invention is to diagnose using methods that allow evaluating the tical set for use in the described analy- structure of proteins, including NMR, circular with which includes a certain amount of non-pathogen- dichroism, Fourier-transformed infrared conformer and, optionally, additionally spectroscopy, Raman spectroscopy, microtiter plate and multiwell ul- sation, internal fluorescence, UV sound device. absorption, etc. Using the method according to wine-

Monoclonal antibody, which is most often possible to detect from 17 to 10 fg patho- is used, against RgR is "ZE4" (Kazszak ei gene conformer, which is initially present in AI., 1987), which is obtained from mice immunized with a sample that is equivalent to From up to 30x1020mol. 262K hamster RiRgase (resistant to proteases The sample will usually be a biological conformer). The specified antibody is also able to bio-recognize a non-pathogenic conformer from a sample or tissue, and any specified bio-recognize a non-pathogenic conformer from a brain sample or tissue can be examined in the brain of a hamster and a human, but not a bovine, using the method according to the invention. In livestock, mouse, rat, sheep or rabbit; it is also a tissue fall, analysis and method according to the judge- able to bind to the pathogenic conformer of the course can be performed on homogenates or without human, but only after denaturation of the specified medium on ex mimo samples. The specified methods and protein. tests will usually be performed on samples

Such antibodies can be labeled to ensure immobilization or immobilization. Preferably, when the sample is baked, easy detection of the marker. For example, some biological fluids such as blood, lymph, urine, scientists use time measurements fluo- or milk; brain tissue, spinal resence using europium-labeled an- brain, tonsillar tissue, or appendix tissue; titila ZE4 | Zaiag eyi a!., 19981. a sample obtained from blood, such as hemolyzed

The methods of detection described above can be used - erythrocytes or leukocyte film preparations; test for the detection of other pathogenic or preparation of plasma membranes, such as conformers, for example, pathogenic forms of Bv- fat mass, resistant to detergents membrane or amyloid protein, and vice versa. caveolae-like domains. Alternatively, a sample

In an alternative embodiment, this can be a composition that includes a non-pathogenic compound (especially a protein) added in an excessive amount in the invention, the conformer obtained in humans can be labeled and can be sub- or animal, such as growth or tissue hormone can be detected , so that the amount of extract, such as pituitary extract. Such a composi- unaggregated conformer until the end of the study tion of the sample may be contaminated with pathogenic will allow to determine the amount of pathogenic confo- conformer. rmera, which was initially present in the sample. The sample may also include food

According to another alternative variant, the product or drink, or a part of the food product of the embodiment of this invention, a pathogenic con- or drink (former (marker) intended for human consumption could be detected by animals), in order to establish the presence of antibodies directed against it. or the absence of a pathogenic conformer in this

In a broader aspect, you can add mi- products or drinks. tissue or a tagging fragment to the pathogenic conformer. It is preferable that the non-pathogenic conformer, former, non-pathogenic conformer or anti- added at stage (i) was of the same species as the one that killed one of the conformers, depending on from school It can be obtained, for example, from the nature of the analysis being performed. moat (i.e., non-pathogenic) form (for example,

Another purpose of this invention is the analysis of the tissue) of the biological sample to be analyzed. the presence of a conformational disease marker. Alternatively, a non-pathogenic conformer can be characterized by a conformational transformation obtained by synthetic or recombinant protein, which lies at its base, with a non-pathogenic pathway, using means known phage into a pathogenic conformer, in the sample; pointers to herders. The analysis includes the following stages: It should be understood, however, that the non-pathogenic contact of the specified sample with some conformer does not necessarily have to be in the pure quantity of the non-pathogenic conformer; or even in an almost pure form. In the majority of cases, the disaggregation of any aggregates that form a non-pathogenic conformer will be in the final result during stage (1), and the tissue homogenate or its fractions, which contain the indicated non-pathogenic conformer. These examples include homogenates of the main types of diseases are prion encephalopathies, including the brain and fractions derived from it, for example fatty spongiform encephalopathy of cattle (B5E) and its human equivalent, disease

Predominantly, the sample and/or non-pathogenic Creutzfeld-Jakob (CO) confobu, in which the protein, rmer will come from a person or the domestic animal underlying them, is RgR. animals such as cows, sheep, goats or cats. The term "sporadic SOJ", abbreviated "500",

Another goal of this invention is to create a compound that modulates Creutzfeldt-Jakob (CO) bonds. This disease-forming transformation of the protein, which lies in the bathroom, is observed spontaneously in individuals at the basis of the disease, from a non-pathogenic to a pathogenic average age of approximately 60 years, with a frequency of 1st conformer, which includes: one case per million people per year worldwide. (І) contact of a certain amount of a non-pathogenic conformer with a certain amount of a pathogenic conformer. compounds; we. The most notable example of such (ii) is the disaggregation of any aggregates that form- the disease is an accidental infection of children, as a result, during stage (ii); by prions from contaminated preparations (ii) determination of the amount of pathogenic confor- human growth hormone. mayor in the presence or absence of the specified The term "family SOI" refers to SOI, which compounds. is rarely observed in families and inevitably causes

If desired, stage (i) may include mutations in the human prion protein gene. stage (ia), during which incubation occurs. The disease occurs as a result of an autosomally specified sample/nonpathogenic conformer and a dominant disorder. Family members who inherit the cycle between stages (ia) and (ii) as having the indicated mutations die from SOY. described above for methods and assays according to the The term "Hertzmann-Strassler- disease of the invention, and vice versa. Scheinker", abbreviated "z55", refers to the for-

If the amount of a pathogenic conformer, we can identify a hereditary human prion disease. wound in the presence of the specified compound, more than the specified disease is observed as a result of the amount measured in its absence, this means that an autosomal dominant disorder. Members of the family, this compound is a factor that "catali- that have inherited a mutant gene, die from 555. zue" conformational transformation; if the specified The term "prion" will mean the transmissible part less, it means that the given compound of the phenomenon, which is known to cause a group of the specified is a factor that inhibits the specified transmissible conformational diseases encephalopathy) in humans and animals. Term

According to the method described above, "iden- "prion" is a contraction of the words "protein" and tification" should also be interpreted as "Infection", and the particles are composed mainly if the "screening" of a number of compounds. not exclusively, from PgRes molecules, the "Label" or "labeling part" can represent Prions are different from bacteria, viruses and are any compound that is used as roids. Known prions include the following prions, a protein detection tool. A tag or part that mi- that infect animals, causing 5sgarie (coagulates, can be attached to a protein by means of becs), a transmissible degenerative disease of ionic or covalent interactions, hydrogen bond- nervous system of sheep and goats, and spongiform zku, electrostatic interactions or intercalation. bovine encephalopathy (B5E) or

Examples of labels or labeling moieties include rabies and spongiform encephalopathies, including, but not limited to, fluorescent conjugates. The following four prion diseases are known to affect humans: (1) kuru, (2) Kreitida disease, chemiluminescent substances, enzymes and Zfeld-Jakob disease (JD), (3) Hertzmann disease. receptors, in such a way that the detection of labeled Strassler-Scheinker (555) and (4) fatal si- protein occurs by fluorescence, konumein insomnia (ER). The term prion, used with streptavidin and/or avidin, quantified in this document, includes all determinations of the radioactivity or chemiluminescence form of prions that cause all or any of the cation, catalytic and/or ligand-receptor interactions - specified diseases or other diseases in action. The preferred label is a fluorescent or any animal and, in particular, human and village-phosphorescent label. farm animals

The term "conformational diseases" refers to the term "PP gene" and "prion protein gene" is used to refer to a group of diseases that arise in this document as an interchangeable trace of the spread of an aberrant conformational way to describe the genetic material that ex- transformation of the protein, which is at their core, which suppresses prion proteins and polymorphisms, and also leads to protein aggregation and its deposition in the tation, such as are listed in this document under tissues. The indicated diseases can also be sub-titled "Pathogenic mutations and polymorphisms - transmitted by means of an induced conformation". The PgiR gene can be from any animal, maternal change, spread from the pathogenic-including "host" and "experimental" conformer to its normal or non-parents described in this document, and be a gene conformer, and in in this case, any and all of its polymorphisms and mutations are present; recognition documents are called "transmissive conform-

but that these terms include genes that ERNIE - fatal familial insomnia; is not a P'P genome, and which have yet to be discovered. 255 - Hertzmann-Strassler disease-

The term "PgR gene" refers mainly to Sheinker; to any gene of any species that encodes PgR' - prion protein; any form of amino acid sequences of PgRe is a normal, non-pathogenic conformer

PgR, including any prion protein. Some RPGs; well-known sequences of RgR (described in the work of Rgrz: - pathogenic or "burnt" - isoform of RgR sabgieva! ei ai., 1992), included in this document as (which is also a marker of prion diseases). links for the disclosure and description of the specified sequences Pathogenic mutations and polymorphisms. There are a number of known pathogenic mutations in the gene

Abbreviations used in this human RgR. In addition, there is a known polymorphism in human, sheep, and bovine RgR genes.

Central nervous system (CNS) - central nervous system; livestock

VE - large spongiform encephalopathy The following is a non-limiting list of cattle; the specified mutations and polymorphisms:

SU - Creutzfeldt-Jakob disease;

Table of mutations

Insertion of 2 octarepeats |Codon 129 Codon 171 5 or 6

Insert 4 octaves | MeiMai Aha/Six octaves

Insertion of 5 octarepeats | Codon 219 (codon 136

Insertion of 6 octave repetitions |SISH/ uz Aa/Mai

Insert 7 octaves

Insert 8 octaves

Insert 9 octaves

Codon 102 of Rgo-th ei

Codon 105 of Rgo-th ei

Codon 117 AIa-Mai

Codon 145 stops

Codon 178 Azr-Avp

Codon 180 Ma!-Pe

Codon 198 Rpe-5eg

Codon 200 cSync-I uz

Codon 210 Ma!-Pe

Codon 217 Avzp-Aga

Codon 232 Mei-AIa

The normal amino acid sequence of the PIR gene of the Syrian hamster (published is observed in the vast majority of individuals - Soidtapp, 1990). The sequence of the PgR gene in mice is called the wild-type PgR sequence. cattle (published by Soidtapp ei

The indicated sequence of the wild type undergoes certain a!., 19911. The sequence of the chicken RgR gene (published characteristic polymorphic changes. In the case of human Naitib5 ei ai., 1991). The sequence of the RgR gene, two polymorphic amino acids of the mink (published Kgei75sptag ei ai., 19921. They follow the residues 129 (Mei/Mai) and 219 (SiIsh/I uv). icedness of the human Ri? gene (published

Sheep RgR has two amino acid polymorphisms according to Kge (25 sptag ei ai., 1986). The sequence of the PgR gene by residues 171 and 136, while the mouse PgR (published by Gospi ei ai!., 1986). Sequence cattle has five or six repeats of the P'gR gene sequence of sheep (published by Uueziaulau ei ai., eight amino acids in the amino-terminal part of the mature prion protein. 19941. The publication data are included in this document. Despite this , that as a reference for the disclosure and description of the PgR gene and am- these polymorphisms in themselves are not pathogenic, but the acid sequence of PgR. they seem to affect prions. This invention also relates to the method of the disease. Certain mutations have been identified to detect the presence of the pathogenic form of the prion gene of human PgIR, different from the indicated normal protein in the sample (mainly in the blood sample or naked variations of wild-type prion proteins, which are exchangeable in the brain), which includes: new specific amino acid residues of PgR or () contact of the sample with some number of non- number of octarepeats, and which differ from the pathogenic prion protein; hereditary human prion diseases. (ia) incubation with disease/non-pathogenic prion-

In order to further understand the given protein; above the map, which demonstrates mutations and polymorphisms- (its) disaggregation of any aggregates that form, you can refer to the published sequence during stage (ia); of RgR genes. For example, two or more ravanas of the Sabgs have been revealed and published. ей ай., 1992, RgR genes of the chicken, large pharynx; and then cattle, sheep, rats and mice. Sequence

(iii) determining the presence and/or amount in the sample - Preferably, if stages (i) and (i) form a cluster of pathogenic prion protein. a cycle that repeats itself at least twice before

Another version of the embodiment of this invention is the implementation of stage (ii), most preferably, if it refers to the method of diagnosis of SOS in a patient, stages (I) and (ii) form a cycle that is repeated, which includes: from 5 to 40 times to performance of the stage (its). taking a sample (mainly a blood sample or a blood sample) from a patient; the presence of a marker of conformational disease - (i) contact of the sample with a certain amount of bien, which is characterized by the conformational transition of RgRe; the creation of the protein underlying it from non- (ia) incubation of the sample/RhRe protein; togenic into a pathogenic conformer in the sample, with (ii) disaggregation of any aggregates, that is, this analysis includes the following occur during stage (ia); stages: repetition of stages (ia)-(ii) two or more times- () contact of the specified sample with a known substance; and then by the amount of the non-pathogenic conformer; (its) determination of the presence and/or amount of PgP5e in (its) disaggregation of any aggregates that form the sample. occur in the final result during stage (i), i

The present invention also relates to the method (ii) of determining the presence and/or quantity in a sample of detecting the presence of a pathogenic form of the indicated pathogenic conformer. Preferably, the amyloid protein in the sample (preferably in the sample so that stages (iii) and (iii) form a cycle that repeats - blood or brain), which includes: sia at least twice before performing stage (iii). (Ж) contacting the sample with a certain amount of non- This invention also relates to the method of pathogenic B-amyloid protein; identification of a compound that modulates the conformation- (a) incubation of the sample/non-pathogenic/D- conversion of the protein underlying the amyloid protein; disease, from a non-pathogenic to a pathogenic confo- (ii) disaggregation of any aggregates, which form, which includes: occur during stage (ia); () contact of a known number of non-pathogens - repetition of stages (ia)-(ii) two or more early conformers with a known number of pathogenic ziv; and then the conformer in the presence or absence of indication- (and) determination of the presence and/or amount of the compound; of pathogenic β-amyloid protein. (its) disaggregation of any aggregates forming

Another version of the implementation of this invention is believed in the final result during stage (i); refers to the method of diagnosing Alzheimer's disease - (ii) determining the amount of pathogenic confor-gamer in a patient, which includes: measure in the presence or absence of the specified taking a sample (mainly a sample of blood or brain compounds) from the patient; The present invention is described with reference to (Y) contacting the sample with some amount of non-specific embodiments of the present invention, but pathogenic B-amyloid protein; the content of the description includes all modifications and for- (a) incubation of the sample/non-pathogenic/D-min, which can be done by a specialist, without amyloid protein; dull from the meaning and purpose of the claims. (iii) disaggregation of any aggregates introduced during stage (ia); by the following examples, which should not be interpreted as repeating stages (ia)-(ii) two or more times as restricting in any way; and then this invention. In the examples, there will be determinations of the presence and/or amount in the sample, referring to the figures described below in this document of the pathogenic β-amyloid protein. documents

The present invention also relates to the apparatus of Fig. 1. Schematic representation of the transformation for use in the methods described above, RIRE PP, especially, to the apparatus, which includes microti- The infectious unit of RgR:: is an oligomer with - th device and some amount of non-pathogenic converts RgRU by including it in the growing former. aggregate, where it acquires properties associated with

Another version of the embodiment of this invention is shown, related to the method of diagnostic detection of Fig. 2. Diagram depicting the procedure of cyclic conformational disease, which is characterized by amplification. The system is based on cycles of conformational transformation of the protein, which is the incubation of RgRze in the presence of an excess of RgRU, from a non-pathogenic to a pathogen - with subsequent cycles of ultrasonic exposure. Under the conformer, by analyzing the sample for the presence of incubation periods of oligomeric PgR, the increase of the marker of the specified disease; is indicated in the size due to the inclusion of PgR in an incremental manner includes: a melting aggregate, and during ultrasonic exposure () contact of the specified sample with a known aggregates are destroyed to increase the amount of non-pathogenic conformer by one; converting bows. In the presented Fig. show (ii) the disaggregation of any aggregates resulting from two cycles of ultrasonication affecting the final result during stage (1) and vu/Iincubation. (ii) determining the presence and/or quantity in the image - Fig. 3. Amplification of PgRe using ku cycles of the specified pathogenic conformer. ultrasonic exposure. A small amount of the homogenate of the affected burned brain, which mis-

tive RgrZe, incubated with the homogenate of the main unit of the centrifuge tube. 7 ml of 3595 sucrose was layered on a sample of a healthy rat (lane 1, control experiment). 1.5 ml of 1595 sucrose was layered on the gradient or with the homogenate of the healthy brain. The test tube is soft (lanes 2 and 3). The last sample was divided into centrifuged at 1500009 for 18 hours. with two groups, one of which was subjected to five cycles of incuba- samples were loaded directly and analyzed for common protein with gel staining and stained for common protein with silver nitrate (panel B) and subjected to Coomassie dye (panel A). The other half was selected by immunoblotting to detect P'P (panel C ). To remove sucrose from the sample, the adipose fraction of the anti-RgR antibody ZE4 (panel B) was prepared for LC and subjected to immunoblotting. Panel C of the masses was isolated, washed in RVZ and centrifuged. It shows some controls in which the homogenate of a healthy person at 28,000 rpm. /min for an hour at 4"C. The brain centers were incubated separately (lanes 1 and 2), the fugue sediment was washed and resuspended in PB5, or in the presence of a diluted lesion homogenate containing 0.590 triton X-100, 0.595 505 and inhibitory 5sgarie of the brain (lanes C and 4) . Half of the proteases. All PgRO was localized in this well of the samples (lanes 2 and 4) subjected to 5 cycles of ultrafractionation (panel 0). sound exposure/incubation. Lanes 2, 3 and 4 of Fig. 8. Factors necessary for amplification were treated with proteinase K. are present in fat masses.

Fig. 4. The sensitivity of the system of cyclic amplifi- Fat masses were isolated from the brain. We studied the minimum concentration of RgR:s, which is a moat hamster, as described in Fig. 2, and mixed with can be detected after amplification, with the help of 700-fold diluted RgRZe high purification by serial dilutions of the homogenate from the affected 5sgarie brain of a hamster "what Zrazzssharie of the brain, and its incubation from the goka were frozen (lane 3) or amplified with the promoter of the brain of a healthy hamster with a draft of 20 hours. (lane 4). Lanes 1 and 2 represent cycles of ultrasonic treatment or without them. The same procedure, but with the use of amplifinel A, shows a control experiment in which uracation of the total brain homogenate. A burned brain of a hamster was subjected to fig.9. Presymptomatic detection of RgRes in serial dilution in hamster brain homogenate. Intracerebral rat in hamsters. Panel B corresponds to the experiment in which physiological solution (cont. serial dilution of the affected 5sgarie holorole group) or diluted 100 times homogenous hamster brain was incubated with the brain of the genat of the affected 5sgarie brain. Every week, a healthy hamster was subjected to 5 cycles of incubation, 4 hamsters from each group were killed, and the main/ultrasonic exposure. The densitometric evaluation of the brain was obtained and homogenized. Half of the immunoblots in A and B are shown in panel C. The samples were immediately frozen (white columns), and the treatment, which was carried out, considering the other half as the original, was subjected to 20 cycles of incubation with brain material, were as follows: 100 ny/ultrasonic exposure ( black bars). All (lane 1), 200 (lane 2), 400 (lane 3), 800 (lane samples were treated with LC and subjected to immunoblotting. 4), 1600 (lane 5) and 3200 (lane 6). The intensity of the bands was evaluated using den-

Fig. 5. Dependence of the RgRhez signal on the amount of cytometry. Each bar represents the average of amplification cycles. Diluted homogenate of ura- value for samples from 4 animals. In the control husband, 5sgarie brains were incubated with brain super-samples, there was no detection, both without an excess of healthy brain homogenate amplification, and with amplification, and the data of the result- hamster. The samples were subjected to 0, 5, 10, 20 or 40 cycles, not shown in Fig. lam, and the PgRhe5 signal was evaluated using Fig. 10. Amplification of human RgRos, Provo- immunoblotting. studies using samples of holo-

Fig. 6. Amplification of RgR:: in blood samples. Parinized rat blood from 11 different confirmed cases of sporadic SOJ was placed in the brain of the brain, as well as from 5 cases of familial damage to the pre-SOI hamster brain and 4 age-matched controls, which included reaching a final dilution of 10:1 . The indicated combination was incubated for 15 minutes in patients suffering from other neurologic diseases. with dark disorders in the room. The brain was homogenized by perature. This material was subjected to 10 times and subjected to 20 cycles of amplification. Representative serial dilutions using heparinized rat blood results from control (A) and three different cases. Samples subjected to 11 cycles of sporadic CO (B) (1, 2, 3) are presented in fig. incubation/ultrasound exposure, and evaluated Fig.11. Detection of RgR:: in the blood after receiving the RgRhez signal using immunoblotting. hemolyzed erythrocytes. Hemolyzed erythema

Fig. 7. Prion protein is present in adipose marocytes from 0.5 ml of heparinized blood of healthy (C) patients. Fat masses (which are also called stable and affected 5sgarie hamsters (5c) were obtained as detergents membrane fraction or OEM) is described in this text. Half of the samples were not isolated, using previously described amplification methods, and the other half were mixed with cola. One hundred mg of brain tissue was homogenized with normal hamster brain homogenate in PB5 medium containing 195 ml of triton X-100 and subjected to 20 cycles of amplification. All samples were filled with a complete cocktail of protease inhibitors (Voengipde?g). therefore, RK was processed and analyzed with the help of immuno

The tissue was homogenized using 10 passages of noblotting. One representative experiment through a 220 syringe needle and incubated is shown in Fig. gm for 30 minutes at 4"C on a rotary stirrer. Fig. 12. Detection of Rgr: in the blood after extraction, the blood was diluted 1:2 in 6095 sucrose and placed with sarkosyl. 0.5 ml of heparinized blood of healthy

(C) and affected 5sgarie hamsters (5c) were subjected to extraction, the amount of common protein, pretreatment with sarkosyl as described in this text. husband in gel, maintained at a constant level

Half of the samples were not subjected to amplification, and the other half (Fig. 3A) with the addition of brain homogenate was mixed with the homogenate of a normal rat to a diluted sample of the damaged 5sgarie hamster brain and subjected to 20 cycles of the brain, using the fact that the RgR of the rat amplification. All samples were then processed by LC and analyzed by the antibody used and lysed by immunoblotting. One re- for immunoblotting. a representative sample from control animals and EXAMPLE 2 two from affected 5sgariye animals are shown in Fig. Detection sensitivity using cyclic Fig.13. Detection of Rgr:: in the blood after isolation and amplification of fat masses. Fat masses were extracted as To estimate the minimum concentration of PgR es, which is described in the text, from 0.5 ml of heparinized blood can be detected after amplification, homogenate of healthy (C) and affected 5sgarie hamsters (5c). One half of the damaged brain was not subjected to amplification, and the other half was mixed with the homogenate of the normal brain of a healthy hamster to swarm dilution directly in the homogenate. Without incubating the hamster brain and subjected to 20 cycles, the PgiRhe5 signal progressively decreased until amplification. All samples were then processed by LC and the anadox did not become such that it was completely undetectable by immunoblotting. One grows at an 800-fold dilution (Fig. 4A, C). a representative sample from control animals and In contrast, when the same dilution was incubated with homo- two from affected 5sgarie animals are shown in Fig. genome of the brain of a healthy hamster and sub-

Fig. 14. Detection of RgR: leukocyte films were obtained in blood after 5 cycles of incubation/ultrasound. The fraction of leukocyte film exposure, the detection limit of RgRhez sharply decreased. blood was separated by centrifugation with 0.5 ml of heparin. Indeed, a clear signal was easily detected even with the nized blood of healthy (C) and affected with 5sgarie 3200-fold dilution (Fig. 4B, C). hamsters (5s). Half of the samples were not subjected to amplification, and the other half were mixed with a homogenate. All samples were then In order to find out whether it depends on the intensity, they were processed by LC and analyzed with the help of the immunoreactivity of the RgRhez signal after cyclic amplification of the loting. One representative experiment from the number of completed incubation cycles is shown in Fig. of ultrasonic/ultrasonic influence, diluted homogenate

EXAMPLE 1 Sections of the affected brain were incubated

Amplification of RgR resistant to RK by dose with an excess of homogenate of the brain of a healthy hamster with cyclic conversion of myocardium. Samples were subjected to 0, 5, 10, 20 or 40

The homogenate of the hamster brain, which was decyclized, and the RgRge5 signal was evaluated using the tala from the affected 5sgarie animals, diluted to dot, immunoblotting. The levels of RgRhez increased exponentially until the signal of RgRos was barely detectable by immunoblotting, depending on the number of cycles of incubation after treatment with proteinase K (RK) (Fig. 3B, confusion/ultrasound exposure (Fig. 5). This result 1) . The processing of RK was carried out with the usual sposotat suggests that an increase in the number of cycles for distinguishing between normal and pathological bones could additionally reduce the limits of the forms of RG'R, which differ in their detection sensitivity. to cleavage by proteases (RgRO: is partially EXAMPLE 4 resistant, and RgRO is cleaved) (Rgivipeg, Experiments with ultrasonic influence on 1991). The form of P'R, which is resistant to the influence of blood samples with a load of Rg

RK, will be further called RgRgez. Incubation of the heparinized rat blood was loaded with a well of the diluted homogenate of the affected 5sgarie brain with the homogenate of the soft brain until reaching the final dilution of 10:1. healthy hamster, which contained an excess of RgRU, was added. A 10-fold serial was made from this material

This suggests that incubation of two dilutions using heparinized blood of brain homogenates leads to conversion. 50 μl of each dilution was centrifuged for RgRU seeds in Rgrz5e, when the samples were incubated in the same at 3000 rpm for 10 minutes. The plasma was separated from the conditions, but subjected to five cycles of incubation and sedimentation. 10 μl of plasma was mixed with nya/ultrasonic exposure, the amount of RgRhev5 increased sharply by 5 μl of homogenate of the brain of a healthy human (Fig. 3B, lane 3). Densitometric test containing the substrate PgR: for the conversion reaction. immunoblot analysis shows that the PgRhev5 signal The samples were subjected to 11 cycles of incubation increased 84 times as a result of cyclic amplification/ultrasonication As a control, the same samples, in comparison with the RgRhe5 signal that was present, were mixed with 50 μl of brain homogenate in a diluted homogenate of a healthy hamster affected by 5sgarie and stored at -20"С until the brain was removed (lane 1).

Conversion depends on the presence of RgRes, oscilloid samples were cleaved with proteinase K, only RgRhez5 was not observed when normal homogenates were separated using 5O5-RACE and analyzed, brain progenitors were incubated separately in those using western blotting, while RgR? was detected conditions, both with and without ultrasonic exposure as described in the "Methods" section. him (Fig. 3C, lane 2). To eliminate artifacts

The results are presented in Fig.b6. The relification data was similar under both conditions. These results show a clear increase in the detection of the results indicate that all the elements that are required for the protein after the amplification procedure, especially the conversion and amplification of PgR (including the so-called obviously at a low concentration of PgR5e (for example, "factor X"; (TeiPpo ei ai ., 1995)), contained in this clade, in breeding 1280). If we compare the data re- specialized membrane domain. Therefore, the results with the results obtained on the tissues, ntification and isolation of factors necessary for the infected brain, then it is possible to obtain PgR conversions, should be possible with the help of confirmation that the amplification process by further separation of proteins from fatty masses by way works in the case of blood. and monitoring their activity using cyclic

EXAMPLE 5 amplification. In addition, fat masses represent so-

Cyclic amplification with high throughput is a possible replacement for the use of total brain homogenate in the procedure of cyclic

The use of traditional ultrasonic amplification as a source of PgR substrate and other devices with a single probe faces the problem of endogenous factors involved in the con- processing of a large number of samples at the same time, as versions. a diagnostic test requires it. EXAMPLE 7 applicants adapted the system of cyclic amplification to ultrasound Presymptomatic diagnostics in an experimental device with a format of 96-well micro- tal animals To study presymptomatic titration tablet (Misopikh 431MR - 20kKGu), diagnostics in hamsters experimentally infected provides ultrasound treatment of all pits of the 5sgarie, the applicants were screened 88 at once and at the same time and can be programmed for brain tumors at different stages of preclinical automatic work. This improvement not only phases, half of which were uninfected counter- reduces processing time, but also prevents loss of role. The brain was sampled every week of material compared to the use of one (4 from each group) and subjected to 20 cycles of the ampliprobe. Cross-contamination is eliminated, ossification. The results showed that this method does not involve direct immersion of the probe into the sample. able to detect pathological protein in the main

The latter circumstance is essential in the analysis of the infected brain even in the second week after inoculation, contaminated samples and minimizes pseudo-positives long before any external results appear in the animals. Twenty cycles of 1 hour. incubation symptoms (Fig. 9). Without cyclic amplification, PgR5e with subsequent ultrasonic pulse exposure was detected in the brain in the sixth week from 15 seconds. to ZbOsek led to reliable after infection, only 4 weeks before the appearance of clinical amplification of the RgRhez signal, similar to the obtained picture of the disease. In none of the control animals previously using the traditional ult- animals that were not infected with 5sgarie, the amplification of the sound device. not found

EXAMPLE 6 EXAMPLE 8

Factors necessary for amplification are found in the detergent-resistant membrane of the human brain. Application of cyclic amplification to samples

The subcellular localization of RgR conversion in patho-specimens of human brains (cadavers) affected by the genesis of the disease has not yet been clarified. However, according to Creutzfeldt-Jakob disease (CJD), the applicants believed that both RgR and Rgrzye located and incubated brain homogenates from several in a special area of the plasma membrane of patients with CJD (or from normal controls) that is resistant to the influence of mild detergents with the homogenate of the brain of a healthy person due to the relatively high content of choleste- and carried out the procedure of cyclic amplification. rin and glycosphingolipids (Meu et al., 1996; Nagteu The results show what is happening dostov- et al., 1995). The specified membrane domains are called for amplification in brain samples that are analyzed with fatty masses or resistant to detergents, with sporadic SOJ, but in none - membranes (OEM), and they contain a large number of mu from 4 control samples amplification of non-repulsive signaling proteins, receptors and ORI-anchor proteins. is carried out (Fig. 10). It is interesting that there was amplification

Applicants confirmed that 10095 RgRU in the main was obtained only in samples that were shown to be brain bound to this fraction, which contains "290 were infected and, thus, able to convert the common protein (Fig.7). Thus, a simple stage of unmutated P'P, while this did not happen, the allocation of fat mass allows a sharp increase when the mutant protein was not able to convert the content of PgPs. Similar results were obtained with the wild-type protein. These data confirm the conclusion of the isolation of fat masses from the homogenate of the affected person that this method works in samples of burnt brain, in which RgRe- was isolated from human fat masses, just as it was shown. previously for samples of animal origin.

In order to evaluate whether the factors, EXAMPLE 9 necessary for the amplification of PgR, are contained in fat masses, Diagnostics by blood by means of cyclic applicants isolated them from the brain of healthy amplification animals and added extremely small amounts Infectivity studies suggest that

High-purity RgRe:s extracted from the brain, at least in experimental animals, Rgres of the brain of sick animals. Amplification in fatty masses present in the blood of animals at the late stage of the disease was equivalent to the amplification obtained by of blood with the help of cyclic amplification, since the amount of RgRhe5 formed after amplification, the applicants preferred to first selectively concentrate the sample for protein, which is under 4"С. - blood proteins such as albumin or hemoglobin were centrifuged at 28,000°C/min for an hour at 4°C. The sediment was washed and resuspended in RV5,

The following four different protocols were shown to contain 0.590 Triton X-100, 0.595 505, and inhibi- were effective in achieving the stated goal. tory proteases. 15 μl of the obtained suspension was mixed 1. Obtaining the shadows of erythrocytes were washed vol./vol. with 1095 brain homogenates

Heparinized hamster blood was centrifuged from a healthy hamster and subjected to 20 incubation cycles at 2500 rpm. at 4"C. Plasma and cell fracturing/ultrasonic exposure. 20 μl of the treatment was separated and frozen and stored with ultrasound and control samples split at 80"C until use. About 0.5 ml of blood cell fractions were digested with proteinase K, separated using washed 3 times in 12-15 volumes of fresh cold 5O5-RACE and analyzed using western РВ5, рН7.6. Cells were resuspended in 12-15 blotting, and PgRZc was detected as described in the section "Methods" in volumes of 20mMOSM sodium phosphate buffer, pH 7.6. The detection results show that Rgray was slightly stirred for 20 min. on ice, after the amplification procedure in blood samples from then centrifuged at 30,000 rpm. during infected animals (Fig. 13). In blood samples from 10 min. at 4"C. The supernatant was removed, the sediment was washed from uninfected animals after signal amplification

With times 20mMOSM sodium phosphate buffer. The end was not observed. Without amplification, the sediment was resuspended in РВ5, which contained 0.590, it was impossible to detect the presence of PgRze (Fig. 13). triton X-100, 0.595 505 and protease inhibitors. 4. Preparation of leukocyte film 15 μl of the obtained suspension was mixed vol./o6. Heparinized hamster blood was centrifuged with 1095 healthy hamster brain homogenate at 1500 rpm. at 4"С for 10 min. Leukocytes and subjected to 20 cycles, the incubation film was carefully isolated using nya/ultrasonication. 20 μl of treated standard procedures and stored at -807С until use. Frozen leukocyte film with proteinase K , were separated using 505- and resuspended in RV5 containing 0.595 triton X-

RACE and was analyzed using western 100, 0.595 505 and protease inhibitors. 15 μl of oder-blotting, and Pgr5c was detected as described in the section of the prepared suspension was mixed vol./0b. with 1095 homo- "Methods". The results show the detection of RgRs in the genome of the brain of a healthy hamster and after the amplification procedure in the blood samples infected with 20 cycles of incubation/ultrasound of forged animals (Fig. 11). Non-infectious effects in blood samples. 20 μl each of ultrasound-treated and convulsed animals, after signal amplification of non-spore control samples, were digested with proteinase K, triaged. Without amplification, it is impossible to detect was separated using 505-RACE and analyzed for the presence of Pg: (Fig. 11). with the help of western blotting, and PgP5e was detected 2. Sarkosyl extraction as described in the "Methods" section. The results of the show-

Heparinized hamster blood was centrifuged to detect RgR: after the amplification procedure at 2500o6./min. at 4"C. 0.5 ml of the fraction of blood cells in the blood samples of infected animals (Fig. 14). In a dilution (06./06.) 2095 was diluted with sarkosyl and incubated in the blood of uninfected animals after amplification for 30 minutes. The sample was centrifuged, no signal was observed. Without amplification, a non-ultracentrifugation Vesktap background was used to clearly detect the presence of PgR ee (Fig. 14). 8a5000o06./min. for 2 hours at 4"C. The precipitate was washed and resuspended in РV5 containing 0.595 tri- Preparation of brain homogenates. a ton of X-100, 0.595 505 and protease inhibitors. 15 microliters of the brain of Syrian golden hamsters, the obtained suspension was mixed vol./06b. from 1095 healthy or infected with the adapted strain, homogenate of the brain of a healthy hamster and burned at 263 K, obtained after decapitation and subjected to 20 cycles of incubation/ultrasound but frozen in dry ice and stored under - exposure. 20 μl each treated with ultrasound and con- 80"С before use. The brain of the homogenized samples was digested with proteinase K, washed in РВ5 with 1095 protease inhibitors (w/v). separated with 505-RACE and analyzed. Detergents were added (0.590 triton X-100, 0.0595 using Western blotting, and RgRZ- was detected by OB) and illuminated by centrifugation on small as described in the "Methods" section. : after the amplification procedure Obtaining samples and cyclic amplification. in blood samples from infected animals (Fig. 12). In Directly in the homogenate of healthy brains, blood samples from uninfected animals after brain amplification, serial dilution of the homogenate was made, no amplification of the signal was observed. Without amplification - the affected 5-burned brain. It is impossible to detect the presence of Rgrzs by ZOmcl odecation. bagging Every hour, a cycle of ultra- 3. Extraction of fatty masses of sound effect (5 pulses per second each) with

Heparinized hamster blood was centrifuged using an ultrasonic microdevice at 2500o6/min. at 4"C. 0.5 ml of the blood cell fraction with a needle dipped in the sample. The specified cycles were re-diluted (0 vol./06.) РВ5 with 195 triton X-100 and stirred several times (5-20). incubated for 30 minutes at 4"C. The sample Detection of Rgre- was diluted 1:2 6095 with sucrose and placed on the bottom of the Samples were split using LC in a centrifuge tube. For a sample, carefully concentrate 100 μg/ml for 90 minutes. at 37"С. 7 ml of 3595 sucrose were re-layered. The gradient was stopped with 50 mm of PM5BE. The samples were separated by layering 1. ml of 1595 sucrose. The test tube was centrifuged with 505-RACE (in denaturing conditions) at 150000°06./min for 18 hours and subjected to electroblotting through nitrocellulose

well membrane in CAP5 or tris-glycine trans- Napteu 9.N., Osuie O., Vgomp M., apa Vodegv ferric buffer with 1095 methanol for 45 min. M.5. (1995). Tne seiIshag isotyugt oi She rgop rgoieieip, at 400mA. Reverse staining of the common RiPs, with avzosiai m/y same oiae ip about this protein, was carried out before blocking the membrane of peigobiasiot (Maga) sei. Viospet. Viorpuz. Nev. 595 skimmed milk. After that, the membrane of Sott. 210, 753-759. incubated for 2 hours. with monoclonal an- Naitiz vy ai., Rhos. May! Asai. 5 BOTH 88:7664- titer ZE4 (1:50000). Washed four times at 7668 (1991). 5 min. РВ5, 0.395 tween 20, before incubation with a secondary anti-mouse antibody labeled with perok- U. (1997). Oiadpoviz oi pej/ mapapi Steshi-leiy UChakor with horseradish sidase (1:5000) for an hour. After pro-disease Bu (opzvii Bbiorvzu. I apsei 349, 99-100. washing, the reactivity in the membrane was developed according to Nvisp a, Keppeu K., (iro5 S.9., 9g., ee K.N., using a kit for chemiluminescence ES. apa Nagipdiyuop MS (1996). Tne 14-3-3 Bgaip rgoivip (Ategzpat), according to the manufacturer's instructions. ip segergozripa! Yamid az a taiKeg g igapetivviriye

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Togspia M, Sopep RE.E., Rgyvzipeg 5.V., (1998). Eidni Togspia M., Sonep E.E., OeAptopa 5.., apa Rgyvzipeg rpop vigaiipye pame RiR(Zs) toiesciev m/yy aiikegepi 5.8. (1995). Riop rgoradayop ip tese ehrgezvipd sopioppayopv5. Mai. Mea. 4, 1157-1165. Ppitap apa spitegtis RgR (gapzdepev itriisagez She

Zagdiasoto M., Ztsao! M., Tapo 7., I iIzapii M.R. iptegasiyup oi seYishiag RIiR U apoipeg rgoivip. Sei (1993). bad luck! ygapvaysipd toiIesshev apa diusovui!- 83, 79-90. rpozrpaiau!iyipoviiui-pKey rgoieip5 Yugt a sameoiip- Magiip Meu ei ai. (1996) Rgo. Maya). Asai. 5bi. psp ipvoiSriye sotrieh ip MOSC seiiv., In Seii! Vioi BA, 93, 14945-9.

Atso; 122(4):789-807. Muereg T., ONo M. Vodeteg M, apa 7egig |. zani M., Vaiimzhmip M.A., Teriozhm O.V., Nosa 1., (1997). Oiadpovziv oi SteshilLeiy-dakor aizease apa sirzop V.MU., Vypipdate A.Yi., apa Rgyvipeg 5.V. heyiaiei PpPytap about 5ropdipt epsernayuratiev. (1993). Bigisyiga! zshaieez oi She zsgarie riop rgoievyp Vioted. RpaptasoiNeg. 51, 381-387. ivipd taz zresigoteyiu apa atipo asia zedpepsipd. Meziamau yei ai., sepev Oeu. 8:959-969 (1994).

Viospet. 32, 1991-2002. МНО/ЕМС/201/98.9, an owl! zZyigmeiPShapse,

Ziviiproii V.)., NasKeg 5., Netepaogi a., apa eyi Oiadpoziz apa TPpegaru oyi Nytap Tgapvtizvirie a!. (1996). Assigasu apa geiyariyu oi regiodis vpagr Zropdiyugtt Epserpaiorainpiee: Verog oi a MNO mzhmame sotriehev ip Steshbseidi-Uakoi aiizease. Agsp. Sopzymayop, Sepema, Zmishepapa 9-11 ERershagu

Meigo!. 53, 162. 1998, M/NO.

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