WO2008072503A1

WO2008072503A1 - Method for detection of abnormal prion

Info

Publication number: WO2008072503A1
Application number: PCT/JP2007/073371
Authority: WO
Inventors: Yoshifumi Yamada
Original assignee: Olympus Corporation
Priority date: 2006-12-12
Filing date: 2007-12-04
Publication date: 2008-06-19
Also published as: JP2010043865A

Abstract

Disclosed is a method which can detect an abnormal prion more rapidly compared to a conventional method. In the method, a sample to be examined on whether or not the sample contains an abnormal prion is mixed with a fluorescently labeled normal prion to prepare a sample solution, and the sample solution is exposed to a reaction condition which enables an abnormal prion to convert a normal prion into an abnormal one. Subsequently, the fluorescence intensity of the fluorescent label attached to the fluorescently labeled normal prion in the sample solution is measured. Based on the measured value of fluorescence intensity, it is determined whether the fluorescently labeled normal prion is present in the sample solution in an unmodified form or in the form incorporated in an aggregate. When it is determined that the fluorescently labeled normal prion is incorporated in an aggregate, then it is concluded that the sample contains an abnormal prion.

Description

Specification

How to detect abnormal prions

Technical field

[0001] The present invention relates to a method for detecting an abnormal prion, and more specifically, the presence of an abnormal prion is detected by fluorescence measurement to detect an abnormal prion in an arbitrary biological sample or a sample derived from a living body. This relates to a method for determining whether or not a message is included.

Background art

[0002] Abnormal prions are used in prion diseases (infectious (transmittable) spongiform encephalopathy) such as bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD) in which the brain of an animal (mammal) becomes sponge-like. However, it is a cohesive (forms aggregate) protein observed in the sponge-like tissue. Regarding the mechanism of the development of prion disease, according to the currently most prominent “prion hypothesis”, abnormal prion itself is a pathogen of prion disease, which normally invades the body of the animal and normally enters neurons in the brain or central nervous system The normal prion (glycoprotein with a molecular weight of about 3.3 to 350,000 (33 to 35 kDa)) is converted to an abnormal prion one after another, and the prion converted to an abnormal type is placed at a specific site. In the brain, it is thought to gradually aggregate and accumulate, and in the brain, the aggregated prion causes the tissue to change into a sponge shape (prion disease develops). In fact, a normal prion is gradually converted to an abnormal prion by repeatedly subjecting a mixture of the abnormal prion and the normal prion to the dispersion process using an ultrasonic wave and an ultrasonic wave. (A method for amplifying abnormal prions is called PMCA: Protein misfolding cyclic amplification. For example, see Non-patent Documents 1 and 2). In addition, abnormal prions enter the body of another animal through food or transplanted tissue derived from animals infected with abnormal prions, medical devices contaminated with body fluids of animals infected with abnormal prions, and the like. Therefore, in order to prevent the spread of prion diseases, abnormal forms of prions or their carriers are detected at an early stage (for example, edible cattle in the BSE problem). In other cases, attempts have been made to block the transmission of abnormal prions to other animal individuals to prevent infection with prion diseases. [0003] When detecting an abnormal prion in order to achieve the above-described object, at present, a method using an antigen-antibody reaction is generally employed. For example, in a whole-body test of beef cattle, traditional methods such as ELISA (solid-phase enzyme immunoassay. For example, Patent Document 1) and Western plot method (Immunoblot method. For example, Non-Patent Document 3). In accordance with the detection method of antigen-antibody reaction, an antibody having an abnormal prion as an antigen is given to a tissue sample prepared from a site where abnormal aggregates of abnormal prions such as the brain and spinal cord accumulate in large quantities after the death of an animal. By detecting whether or not an antigen-antibody reaction occurs, it is determined whether or not there is an abnormal prion in the animal body, that is, whether or not the animal is a carrier of the abnormal prion. Further, in recent years, as a detection method that does not use a tissue at a specific site, in the above Non-Patent Documents 1 and 2, an abnormal prion present in a trace amount in the blood of an animal is amplified by PMCA, and then the antigen antibody as described above is used. A method for confirming the presence or absence of a trace amount of abnormal prion in a biological sample such as blood by using a detection method by reaction has been reported. Further, in place of the above-described classical antigen-antibody reaction detection method, in Patent Document 2, an antibody against an abnormal prion is fluorescently labeled, the antibody and the sample to be examined are mixed, and an abnormal prion ( Disclosed is a method for detecting the presence of an abnormal prion in an arbitrary sample by detecting whether or not an antigen-antibody reaction between the antigen and the antibody has occurred by using fluorescence correlation spectroscopy. .

[0004] As a method that can be used for detection of abnormal prions without using the antigen-antibody reaction as described above, in Patent Document 3, a test sample is directly labeled with a fluorescent dye, and fluorescence correlation spectroscopy is performed. Has been proposed to detect the presence / absence of proteins that aggregate and deposit in the test sample (strictly speaking, this method does not detect abnormal prions in the test sample). This is a method for determining whether or not a protein has the property of aggregating o) o

Patent Document 1: US Pat. No. 4,806,627 Specification

Patent Document 2: Japanese Patent Laid-Open No. 2005-345311

Patent Document 3: Special Table 2001—517800

Non-patent literature 1: Saborio et al. 2 people, Nature 411, 810-813, 2001 Non-patent literature 2: Saar et al. 2 people, Science 313, 92-94, 2006 Non-Patent Document 3: Barry and Prussian I, Journal of Infexiyas Diseases 154 518-521, 1986

Non-patent document 4: Lee et al. Genome Res. 1998 8 1022-10 10 37

Non-Patent Document 5: 2 other than bread, etc. Protein 'Science (1992) 1 pp. 343- 1352

Disclosure of the invention

Problems to be solved by the invention

[0005] When detecting abnormal prions on a large scale, not on a laboratory level, for example, for clinical diagnosis or when testing a large number of specimens such as whole-head inspection of beef cattle In such cases, it is desirable that the detection method should be able to be carried out promptly with the ability to test individual specimens with high accuracy. However, in the case of the classic detection method using the antigen-antibody reaction as described above, it takes time until a test result with many processing operations is obtained.

[0006] In addition, in order to detect the presence or absence of a detection target substance with sufficient accuracy by a conventional method using an antigen-antibody reaction, a considerable amount of the detection target substance is generally contained in a test sample. Therefore, even when detecting abnormal prions, it is necessary to prepare a sample containing abnormal prions with a considerably high content. In addition, it is difficult to create an antibody that recognizes both normal and abnormal prions with high accuracy and distinguishes them from each other. It is difficult to prepare an abnormal prion by an inspection method using the antigen-antibody reaction as described above. In actuality, before the antigen-antibody reaction is caused (before the antibody is allowed to act on the test sample), normal prions in the test sample are made normal by protease K or other drugs. When the mold prion is removed, a process is necessary. In such a case, a considerable amount of abnormal prions may be lost during the process of removing normal prions from the test sample. In anticipation of the loss in the sample processing operation, it is necessary to prepare a test sample containing a large amount of anomalous prions (as compared to a detection method using an antigen-antibody reaction of a normal substance). In the case of the above-mentioned method of Patent Document 3, antigen-antibody reaction is not used, but it is determined whether there is a test sample or aggregation. A processing operation is required in which the target substance to be determined is directly labeled with a fluorescent dye, and a considerable amount of the test target substance may be lost during the processing operation.

In other words, the currently available abnormal prion detection methods require many processing operations and a long processing time, and a test sample containing a considerable amount of a test substance, that is, an abnormal prion. Otherwise, it is difficult to obtain a reliable detection result. If there is a method that can detect anomalous prions in a short time than before, even if the amount of anomalous prions in the sample is very small, it will be very advantageous for detecting anomalous prions on a large scale. .

In other words, an object of the present invention is a method for detecting an abnormal prion, which can detect an abnormal prion extremely quickly compared to the conventional method without using an antigen-antibody reaction. Is to provide a method.

[0009] Further, another object of the present invention is to reduce the number of treatments of a test sample in which the presence or absence of an abnormal type prion should be detected or determined, thereby losing the abnormal type prion to be detected during the processing. The present invention also provides a method capable of detecting abnormal prions even when the content of abnormal prions is very small.

[0010] Still another object of the present invention is to provide a method for detecting an abnormal prion as described above, and a method for detecting an abnormal prion using fluorescence measurement / analysis. Means to solve

[0011] According to the present invention, there is provided a method that can advantageously detect an abnormal prion even if the amount of the abnormal prion in a test sample is small, without using an antigen-antibody reaction. The method for detecting an abnormal prion of the present invention is a process of preparing a test sample solution by mixing a test sample to be examined for whether or not it contains an abnormal prion and a fluorescent-labeled normal prion. The process of exposing the test sample solution to reaction conditions in which abnormal prions convert normal prions to abnormal prions, and the fluorescence-labeled normal type in test sample solutions exposed to force and reaction conditions The process of measuring the fluorescence intensity of the fluorescent label added to the prion, and the normal prion fluorescently labeled based on the fluorescence intensity is present alone in the sample solution! / And the process of determining whether it is incorporated in the aggregate. So When it is determined that the fluorescently labeled normal prion is incorporated into the aggregate, the abnormal prion that converts the fluorescently labeled normal prion to the abnormal prion is detected in the test sample. It is determined that it is included!

[0012] According to the configuration of the present invention described above, first, a test sample to be inspected whether or not it contains an abnormal prion is mixed with a fluorescent-labeled normal prion, and the mixture (test sample) Solution) is exposed to reaction conditions that convert it to abnormal prions. As shown in the above-mentioned prion hypothesis or in Non-Patent Document 1! / As shown, if an abnormal type prion is included in the test sample, the normal type prion is changed to the abnormal type prion. As a result, an aggregate of abnormal prions is formed, so that the fluorescently labeled normal prion molecules that were moving independently before mixing with the abnormal prion are also mixed with the abnormal prion after mixing with the abnormal prion. It becomes an abnormal type prion (labeled with fluorescence) and is incorporated into the aggregate.

[0013] Whether or not a prion molecule that is a normal type labeled with fluorescence is converted into an abnormal type and incorporated into an aggregate can be determined through the fluorescence intensity of the fluorescent label added to the molecule. If a fluorescent label is added to a molecule such as a protein as understood by those skilled in the art, changes in the structure, size, and movement of the molecule can be detected based on the fluorescence intensity of the fluorescent label. In the case of the present invention, the prion molecule that has been moving as a single molecule is incorporated into the aggregate and moves integrally with the aggregate. Therefore, the change in movement accompanying the change in the state is added to the prion molecule. This is reflected in changes in the various behaviors (fluctuations) of the fluorescence intensity of the fluorescent label, and whether or not normal prion molecules have been converted into abnormal prion molecules, that is, abnormal prion molecules in the test sample. It will be judged whether or not there existed. In short, the present invention shows that when normal prions are mixed with abnormal prions, normal prions are converted into abnormal prions and incorporated into aggregates of abnormal prions! / It is possible to detect the activity of this by fluorescence measurement, thereby detecting the presence or absence of an abnormal prion.

[0014] When abnormal prions are present in the tissues or body fluids of animals infected with prion diseases as shown in Non-Patent Documents 1 and 2, in vitro, Even in the system, it is known that normal prions are converted to abnormal prions. Therefore, in the above-described configuration of the present invention, the test sample is an animal tissue or animal sample collected from an animal. It may be a body fluid. In particular, according to the findings of Non-Patent Document 2, it is found that abnormal prions present in the blood of animals infected with prion diseases cause a conversion reaction from normal prions to abnormal prions in an in vitro system. Therefore, in the method of the present invention, typically, the force, animal tissue or body fluid sample which is easily collected can be advantageously used as the animal blood or a part thereof. . In addition, it has been confirmed that the conversion reaction from normal prions to abnormal prions proceeds in the presence of animal body fluid samples containing normal prions. Thus, for example, if there is not enough normal prion in the test sample, and it is assumed that the conversion reaction will proceed only with the test sample and fluorescently labeled prion! / It is preferable that an animal body fluid sample containing prions is further mixed into the test sample solution. In particular, animal fluid samples containing normal prions that are added to promote the conversion of normal prions to abnormal prions are samples prepared from brain tissue collected from animals not infected with prion disease. It's okay. However, if it is found in preliminary experiments that the conversion reaction proceeds even in a mixture of the test sample and fluorescently labeled prions, V, if prepared from animal body fluid samples or brain tissues containing normal prions. In such a case, it is not necessary to mix the prepared sample.

[0015] Specific conditions in the process of exposing the test sample solution of the above method to reaction conditions in which abnormal prions convert normal prions to abnormal prions are determined by those skilled in the art according to the method of the present invention. It should be understood that prior to implementation, it can be found by preliminary experiments. The conditions for the conversion reaction from normal prions to abnormal prions include force S that may vary depending on the species of animal S, and the presence of abnormal prions. The conditions for causing the conversion reaction from the normal prion to the abnormal prion can be found by using the test sample and carrying out the process of the present invention. According to the findings of Non-Patent Documents 1 and 2, typically, the reaction condition is to incubate the test sample solution for at least 30 minutes at 37 ° C.

In the above-described method of the present invention, the measurement of the fluorescence intensity for observing the state of the fluorescence-labeled prion molecule is the so-called “single-molecule fluorescence analysis technique”, that is, the fluorescence from one fluorescent molecule. Measure the light and analyze its temporal change or fluctuation to observe the state or movement of a single fluorescent molecule. It can be advantageously carried out by the fluorescence analysis method to be measured. Such single molecule fluorescence analysis techniques include Fluorescence Correlation Spectroscopy (FCS), Fluorescence—Intensity Distribution Analysis (FID A), and Fluorescence Correlation Spectroscopy (Fluorescence). It can be any force implemented by a fluorescence measuring device such as cross-correlation spectroscopy (FCCS) that combines an optical system of a laser confocal microscope with an ultrasensitive photodetection device (by the configuration of the present invention). The results of fluorescence measurement and analysis in each fluorescence analysis method will be explained in the description section of the embodiment). Also, any fluorescence analysis method that can observe changes in the structure, state, and motion of other molecules, such as fluorescence depolarization spectroscopy (FDS), may be used! / (In this case It may be a normal fluorescence depolarization measurement device rather than a device that performs single-molecule fluorescence analysis.) It should be understood that any fluorescence analysis method that can detect whether a certain fluorescently labeled molecule (prion molecule) moves alone! /, Or forms an aggregate. Such cases are also within the scope of the present invention.

The invention's effect

[0017] As understood from the above description, the present invention uses a normal-type prion fluorescently labeled as a probe, detects the phenomenon that the probe is converted to an abnormal-type prion, and detects the abnormal-type prion. The antigen-antibody reaction is not used to detect the presence of abnormal prions. Therefore, it is not necessary to produce an antibody that recognizes and distinguishes between abnormal prions and normal prions accurately, and also does not require treatment to remove normal prions from the test sample (in the present invention, It is desirable that normal prion is present in the test sample because it detects the conversion reaction from normal to abnormal form of prion.) In the conventional detection method using antigen-antibody reaction, Various problems caused by the difficulty in producing antibodies that specifically recognize abnormal prions can be avoided.

[0018] In the present invention, it is necessary to prepare a fluorescently-labeled normal prion in advance, but the processing in the examination of individual specimens is carried out with the specimen and the fluorescence. Mix with the labeled normal prion and maintain the conditions under which the fluorescent labeled prion initiates the conversion reaction. After that, it is only necessary to perform the fluorescence measurement, so that it can be completed in a much shorter time than before. Furthermore, in the present invention, there is no need to perform a process for chemically modifying substances in the test sample, such as a process for removing normal prions from the test sample or a process for fluorescently labeling the test sample. Therefore, the abnormal prion that is the detection target in the test sample during such processing is not lost during the processing operation. Therefore, the abnormal prion is lost during such processing. This eliminates problems such as false negative results. Furthermore, according to the aspect of detecting a reaction in which a fluorescent-labeled normal prion is converted to an abnormal prion by a fluorescence measurement without chemically modifying the test sample, the test sample is included in the test sample. Therefore, it is expected that the presence or absence of abnormal prions contained in a small amount of animal body fluid can be determined. In other words, in order to obtain a test sample, it is not necessary to collect tissue in which abnormal prions such as the brain and spinal cord of animals are accumulated in large quantities or to amplify abnormal prions by PMCA.

[0019] In summary, according to the present invention, an abnormal prion can be detected in a short time using a trace amount or a small amount of a test sample. In the present invention, the test sample is a sample that can be collected in a minimally invasive manner, such as animal blood! /. It is expected to be used for large-scale tests such as the determination of type prion carriers or the inspection of all beef cattle.

[0020] Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

Brief Description of Drawings

[0021] [FIG. 1] FIG. 1 is a flowchart showing the processing steps in a preferred embodiment of the method of the present invention.

[FIG. 2] FIG. 2 is a schematic representation of changes in the state of prion molecules during the process of the embodiment of FIG. 1 when abnormal prions are present in the test sample. . (a) is a fluorescently labeled normal prion, (a ′) is a body fluid sample containing a normal prion optionally added, and (b) is a test sample. When the sample solution (c) prepared by mixing (a), (a '), and (b) is exposed to the condition (d) where the conversion reaction occurs, it is shown in the lower figure of (d). Normal prions as shown above are converted into abnormal forms to form aggregates (e).

[FIG. 3] FIG. 3 is a view similar to FIG. 2 in the case where a normal prion to which two different fluorescent labels are separately added is used as a probe.

BEST MODE FOR CARRYING OUT THE INVENTION

[0022] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

[0023] FIG. 1 is a flowchart showing the processing steps in a preferred embodiment of the method for detecting anomalous prions of the present invention, and FIG. 2 is assumed in the processing of FIG. The change of the state of the molecule is schematically shown. Referring to the figure, in the test according to the method of the present invention, first, a fluorescent-labeled normal prion and a test sample are mixed to prepare a test sample solution (step 10).

[0024] The fluorescently labeled normal prion (FIG. 2 (a)) used as a probe is prepared in advance by any known method for those skilled in the art (see the Examples section). The normal prion is typically extracted by any known method for a person skilled in the art from tissue of an animal uninfected with a prion disease of the same species as the animal to be examined, such as the brain and spinal cord. It may be. In addition, the fluorescent labeling of normal prion may be performed, for example, by adding a fluorescent molecule to the prion by a chemical labeling method targeting a specific group in the prion after preparing the normal prion. The fluorescent dye may be any optical dye usually used in this field, such as TAMRA (carboxymethylrhodamine), TMR (tetramethylrhodamine), Alexa647, Rhodamine Green, Alexa488, but is not limited thereto. When performing fluorescence measurement as described later with FIDA, it is desirable that the number of fluorescent labels (fluorescent dye molecules) added to each prion molecule is the same. When the fluorescent labeling is performed by the chemical labeling method, the fluorescent dye may be a fluorescent dye reagent for chemical labeling fluorescent labeling such as NHS ester or maleimide. In addition, if possible, the preparation of fluorescently labeled normal prions adds fluorescent dyes to specific groups more accurately in the cell-free protein synthesis system using intracellular prion cDNA. Using a pinpoint fluorescent labeling technology that produces proteins and a method to prepare normal prions fused with fluorescent proteins such as GFP And may be labeled as normal prions. Furthermore, when the fluorescence measurement described later is performed by FCCS, at least two normal prions labeled with different fluorescent dyes are prepared in the same manner as described above. Fluorescently-labeled normal prions can be stored in any way so as not to denature, in which case only the amount required for the test should be used.

[0025] The test sample, that is, the sample to be inspected whether or not the abnormal prion is contained (FIG. 2 (b)) is a substance that may contain the abnormal prion, for example, an animal. It may be any of tissue such as brain, spine and the like, body fluid such as blood and urine, food, and tissue for transplantation. When determining whether or not a certain animal is infected with prion disease or whether it is a carrier of abnormal prion (early diagnosis of prion disease), the blood of the animal may be used. In this regard, since prions are known to exist in a buffy coat obtained by centrifugal fractionation of blood, only such buffy coat is preferably used when blood is used as a test sample. Is done.

[0026] Further, the test sample solution is preferably a tissue of an animal uninfected with a prion disease of the same kind as the animal to be examined (for example, a brain, spinal cord homogenate solution, etc.) (fluorescence A bodily fluid sample (Fig. 2 (a ')) obtained from a living body containing a large amount of normal prion (not labeled) is mixed. Although the exact reason has not been elucidated, it has been reported that abnormal prions convert normal prions into abnormal prions in body fluids containing a large amount of normal prions such as brain and spinal cord homogenates. (For example, Non-Patent Document 2). Therefore, if it is assumed that the conversion reaction does not proceed only with the test sample and the fluorescently labeled normal prion, the conversion reaction is further performed by adding a body fluid containing a large amount of the normal prion as described above. The condition for the process to proceed may be satisfied. If the conversion reaction is considered to proceed even with the test sample alone, or the fluorescently labeled normal prion is a fluorescent protein fusion prion expressed in an animal by genetic recombination, etc., and an abnormal prion is mixed. If so, body fluids containing large amounts of normal prions (not fluorescently labeled) may not be mixed if the conversion reaction is thought to proceed.

[0027] The test sample solution prepared in Step 10 (FIG. 2 (c)) is exposed to a predetermined condition in which the conversion reaction proceeds for a predetermined time (Step 20). Non-speciality listed in PMCA! / According to the knowledge in Permissible Literatures 1 and 2, when the animal is a hamster, incubation at 37 ° C for about 30 minutes converts some normal prions into abnormal prions (Fig. 2 (d )), Found to form small abnormal prion aggregates! /, (The exact mechanism has not been elucidated. It is thought that the conversion reaction will be slowed if the aggregates are larger than if all of these are converted to abnormal forms at once.) It should be noted that the predetermined conditions and the predetermined time for proceeding with the conversion reaction described above are preliminarily performed by a person skilled in the art using a test sample that is known to contain an abnormal prion, and the fluorescent labeling is performed. It should be understood that it can be determined by finding the conditions under which normal prions are converted to abnormal forms.

[0028] In step 20, if the test sample contains an abnormal prion, the normal type fluorescently labeled as shown schematically in FIG. 2 (e). Some normal prions including prions are converted into abnormal prions to form aggregates. When the fluorescently labeled normal prion that was moving alone before mixing with the test sample is incorporated into the aggregate, the state of the Brownian movement of the fluorescent label added to the prion molecule changes. . Therefore, the fluorescence intensity of the sample solution exposed to the predetermined conditions under which the conversion reaction proceeds in step 20 can be measured by any measurement / spectroscopic analysis method that can detect the change in the movement state of the fluorescent label of the prion molecule. Measurement and analysis are performed (step 30). The fluorescence intensity measurement method adopted here is, for example, FCS, FDS performed using a fluorescence measurement device (single molecule fluorescence analysis device) that combines an optical system of a laser confocal microscope with an ultrasensitive photodetection device. , FIDA, or FCCS.

[0029] In FCS (Fluorescence Correlation Spectroscopy), the speed of movement (translational motion) of molecules or particles passing through a minute fluorescence observation region (the focal region of an object lens of a laser confocal microscope) by Brownian motion is observed. Is done. The speed of translation of the molecule or particle is reflected in the shape of the autocorrelation function with the time of the measured fluorescence intensity as a variable. As an index of the speed of translation of molecules or particles, the length of time (translational diffusion time) from the start of measurement until the autocorrelation function value is halved is used. Since the movement of a molecule or particle becomes slower as its size increases, the translational diffusion time becomes longer. In the case of the present invention, the normal type prion molecular force that had been in Brownian motion alone as shown in Figs. 2 (a) and (c) was converted into an abnormal type as shown in Figs. 2 (d) and (e). When the aggregate is formed, the fluorescent label added to the prion molecule is constrained on the aggregate and moves integrally therewith. Therefore, compared to the cases of Fig. 2 (a) and (c), Since the speed of movement of the dye is remarkably reduced and the length of the translational diffusion time is increased, it is detected whether or not the fluorescently labeled normal prion molecule is converted into an abnormal form and aggregated.

[0030] In FDS (fluorescence depolarization method), the speed of rotational browning (rotation) of molecules as known in this field is observed. The speed of the rotational movement of the molecule is reflected in the measured ratio of the intensity of longitudinally polarized light to transversely polarized light or the degree of polarization. Since the rotation of the molecule becomes slower as the size of the molecule increases, the degree of polarization increases. In the case of the present invention, when the fluorescently labeled prion molecule as shown in FIG. 2 (e) forms an aggregate, as in the case of FCS described above, the fluorescent label is bound to the aggregate. ) And (c), the rotational speed of the fluorescent label is significantly reduced and the degree of polarization increases, so that the normal prion molecule labeled with fluorescence is converted into an abnormal form and aggregated. It is detected whether or not.

[0031] In FIDA (fluorescence intensity distribution analysis method), photons emitted from a minute fluorescence observation region are detected (photon counting), and the frequency of detection of photons per unit time is statistically processed. Thus, the number density of fluorescent particles in the minute fluorescent observation region and the fluorescent intensity per fluorescent particle are calculated. In the case of the present invention, fluorescently labeled prion molecules form aggregates. In particular, a plurality of fluorescent labels are bound to one aggregate such as the aggregate α in FIG. When moving as one fluorescent particle, the number density of fluorescent particles is reduced, while the fluorescent intensity per fluorescent particle is increased due to the presence of multiple fluorescent labels in the aggregate. Therefore, from the decrease in the number density of the fluorescent particles and the increase in the fluorescence intensity per fluorescent particle, the force and power that are detected when the fluorescently labeled normal prion molecules are converted into abnormal forms and aggregated are detected. Become. In this case, in order to detect the presence or absence of an increase in the fluorescence intensity per fluorescent particle with high sensitivity, as described above, the fluorescence-labeled normal prion is added to each prion molecule. It is desirable that the number of fluorescent labels to be fixed is constant (because there is a variation in the number of fluorescent labels per prion molecule, it is possible to detect a change in fluorescence intensity per fluorescent particle).

[0032] In FCCS (fluorescence cross-correlation spectroscopy), when two fluorescent labels with different emission wavelengths pass through a minute fluorescence observation region by Brownian motion, the fluorescence intensity of each label changes. It can be determined whether there is a correlation between the movements of the two fluorescent labels. If two fluorescent labels are present on one carrier, the changes in the two fluorescence intensities change together, but if the fluorescent labels are present on different carriers, the two fluorescent intensities Change will change independently. Whether or not a fluorescent label is on one carrier can be determined from the cross-correlation function of two fluorescent intensities with time as a variable. In the present invention, when a prion molecule to which fluorescent labels A and B different from each other are used, the prion molecule carrying the fluorescent label A and the fluorescence as shown in FIGS. 3 (a) and (c) are used. When the prion molecule carrying the label B is incorporated into one aggregate, the different fluorescent labels lA and B move physically, and the prion molecule independently performs a Brownian motion. Since the cross-correlation function is higher than in the case, it is detected whether the fluorescent-labeled normal prion molecule is converted into an abnormal form and aggregated.

[0033] If it is detected by the above fluorescence measurement that the normal prion molecule labeled with fluorescence is converted to an abnormal form and aggregated (step 40), the abnormal prion is present in the test sample. It will be judged that!

[0034] What should be noted regarding the above-described method is that it detects that the fluorescent-labeled normal prion has been converted to an abnormal type, and therefore the abnormal type detected by the method of the present invention. The prion means that an abnormal prion that retains the activity of converting a normal prion into an abnormal form, that is, has a pathogenicity of prion disease, is detected. In this regard, it is possible to detect the presence or absence of an abnormal prion by the conventional detection method using an antigen-antibody reaction or only by detecting the aggregation property of a protein. The detected abnormal type prion is converted to a normal type prion. I can't judge if I have the ability!

[0035] In addition, when an inspection is performed using any one of the above-described series of fluorescence analysis methods using a single-molecule fluorescence analyzer, the amount of sample required to detect one result may be about several tens, The measurement time can be as long as 5 to 15 seconds of measurement is repeated several times, so the sample volume and time can be greatly reduced compared to conventional biochemical methods. In this regard, in the fluorescence measurement as described above, it is possible to detect changes in the state of molecular motion when prions form aggregates of several molecules. Therefore, it is not necessary to amplify the abnormal prion to the extent that it can be detected. It should be understood that the PMCA described in 2 above, ie, the process of repeating incubation and dispersion with ultrasound many times is not necessarily required! The force depending on the test sample It is considered that the conversion reaction treatment in the method of the present invention may be detected only by performing the first incubation in PMCA (of course, the ink of the test sample solution). (Evection and ultrasonic dispersion treatment may be repeated). Furthermore, the method of the present invention does not require chemical modification of the test sample or treatment for removing normal prions from the test sample, so that an abnormal type in the test sample is not measured before fluorescence measurement. The amount of abnormal prions in a detectable test sample that is very unlikely to lose prions is further reduced compared to previous methods.

[0036] According to the method of the present invention described above, any animal for which a tissue- or body-fluid sample containing abundant prions such as a normal prion that is fluorescently labeled and a prion disease-uninfected brain or spinal cord is available. It is possible to detect or determine whether or not there is an abnormal prion.

[0037] Specifically, the abnormal prion detection method of the present invention described above may be performed as follows. In addition, it should be understood that the following examples do not limit the scope of the present invention, force S, which is an example of detection of an abnormal hamster prion. Example 1

[0038] Abnormal prion detection using normal prion fluorescently labeled with one kind of fluorescent dye Preparation of probe sample

Based on the prion base sequence described in Non-Patent Document 4, the fluorescence-labeled normal prion used as the probe sample is labeled with the fluorescent dye TAMRA using the pinpoint fluorescence labeling kit (Olympus). can get. The prepared normal prion for fluorescent labeling is prepared to be ΙΟΟηPBS in dilution buffer (PBS (sigma), 1% Triton X-100, Complete Protease Inhibitor Cocktail (Roche)). May be prepared by chemical labeling after extracting and purifying normal prions as in Example 2 below). In addition, the body fluid containing normal prion is added in a test tube to 200 mg of prion disease-uninfected hamster brain tissue with 800 μl of dilution buffer, and the tissue is made into a uniform emulsion using an ultrasonic crusher. Until sonicated. Then milk The diluted brain tissue solution is diluted to about 1% with a dilution buffer (diluted brain emulsified solution).

).

[0039] Preparation of test sample

The blood lml collected from the test animal using EDTA-containing blood collection tubes (Betaton's Dickinson) is centrifuged at 500 X g for 5 minutes at 25 ° C, and the leukocyte layer (buffy coat) fraction is collected. The collected buffy coat fraction is freeze-thawed 3 times and centrifuged at lOOOOOOg 4 ° C for 1 hour. The precipitate is collected and used as a test sample.

[0040] Conversion reaction

10011 1 diluted brain emulsified solution and 10 1 fluorescent labeled normal prion solution are mixed with the total amount of collected precipitate (test sample) (test sample solution) and incubated at 37 ° C for 30 minutes .

[0041] Fluorescence measurement and analysis

(l) Measurement and analysis by FCS

Dilute the sample solution to be reacted 10-fold with a dilution buffer, and measure the fluorescence intensity of 50 1 samples using the single molecule fluorescence analysis system MF20 (Olympus) according to FCS. The single molecule fluorescence analysis system calculates the autocorrelation function of the measured fluorescence intensity according to the internal program, and calculates the translational diffusion time. When a normal fluorescent-labeled prion is in Brownian motion alone in a solution, the translational diffusion time is about 370 seconds as estimated from its molecular weight (3.3 to 350,000). On the other hand, if fluorescent-labeled normal prions are incorporated into about 50 aggregates, the translational diffusion time calculated from the autocorrelation function of the fluorescence intensity at that time is estimated to be about 1360 seconds. Fortunately, if the test sample contains anomalous prions, and the normal fluorescence-labeled prions are incorporated into several aggregates by the conversion reaction, the translational diffusion time becomes longer and the fluorescence-labeled normal is normal. It is detected that the type prion is converted to an abnormal type and aggregated. That is, the abnormal force prion is included in the test sample! /, And the force S is detected.

[0042] (2) Measurement and analysis by FIDA

Measure the fluorescence intensity of the sample specimen for fluorescence measurement in the same way as above using the single molecule fluorescence analysis system MF20 (Olympus) according to FIDA. Single molecule fluorescence analysis system The fluorescence photon counting is performed according to the program of Fig. 1, and the number density of fluorescent emitters in the solution and the fluorescence intensity per emitter (number of emitted photons per unit time) are obtained from the histogram of the number of received photons obtained using time as a variable. ) Is calculated. When the fluorescent-labeled normal prion is in Brown motion alone in the solution! /, The fluorescence intensity per molecule with the fluorescent-labeled normal prion alone is estimated to be about 35 kHz. On the other hand, if about 4 to 5 fluorescently labeled normal prions are incorporated into one aggregate, the fluorescence intensity of one luminescent particle (aggregate) is estimated to be 126.1 kHz. For example, if the test sample contains abnormal prions, and fluorescence-labeled normal prions are incorporated into the aggregate by the conversion reaction, the fluorescence intensity (number of emitted photons per unit time) is Luminous particles (aggregates) are detected larger than in the case of molecules, and the force is detected that the fluorescent-labeled normal prion is converted to an abnormal form and aggregated. That is, it is detected that the abnormal sample contains a prion.

Example 2

[0043] Extraction of abnormal prions using two types of fluorescently labeled normal prions fluorescently labeled with different fluorescent dyes

The treatment process is the same as in Example 1 until the conversion reaction process, except that two types of fluorescently labeled normal prions are used as probe samples. As a probe sample, a fluorescent labeled normal prion to which rhodamine green (Invitrogen) is added and a fluorescent labeled normal prion to which alexa647 (Invitrogen) is added are used. The fluorescently labeled normal prion may be prepared by adding a fluorescent dye to the normal prion extracted and purified as follows by a chemical labeling method.

[0044] The procedure for extracting and purifying normal prions is as follows (see Non-Patent Document 5).

(1) 900 g of normal hamster brain with 900 g of buffer (sucrose, 0.1 mg / ml TPCK (sigma), lmM PMSF (sigma), lOmM sodium phosphate (pH 7.0), 0.15 M Na CI) In addition, disrupt the tissue using polytron, then centrifuge the sample at 3000 X g for 30 minutes and collect the supernatant.

(2) Add PEG-8000 to 4% to the collected supernatant, stir at 4 ° C for 15 minutes, force, and centrifuge at 1400 X g for 10 minutes to collect the precipitate. (3) The precipitate was added to ZW3-12 buffer (0.15 M NaCl, 10 mM sodium phosphate (pH 7.0), 0.1 mg / ml TPCK, ImM PMSF, 8% ZW3-12 (N-Dodecyl-N, N-dimethyto 3-ammonio- 1-propanesulfonate) (sigma)) and diluted so that the weight of ZW3-12: weight of sediment = 10: 1, and stirred at 4 ° C for 1 hour Centrifuge at 100000 xg for 1 hour and collect the supernatant.

(4) The collected supernatant was adsorbed with copper ions and equilibrated with buffer A (0.15 M NaCl, 10 mM sodium phosphate (ρΗ7.0), 0.2% ZW3-12). Add to a Flow (Chelating Sepharose Fast Flow) column (GE Healthcare Life Sciences).

(5) The column of (4) given the supernatant recovered in (3) was washed with buffer A plus 10 mM imidazole, then 0.5 M NaCl, 0.2% ZW3-12 Wash with 10 mM buffer, and finally with 10 mM sodium phosphate buffer (ρΗ7.0).

(6) When a buffer containing 50 mM EDTA, 0.2% ZW3-12, 10 mM sodium phosphate, and 0.2 M NaCl is applied to the column, normal prions are eluted from the column.

The normal prion used for the preparation of the fluorescence-labeled normal prion may be a commercially available extracted and purified product (for example, Native PrPchamster recombinant manufactured by JENA BIOSCIENCE).

The above-mentioned fluorescent dye is added to the normal prion obtained by the chemical labeling method.

Fluorescence measurement and analysis

The fluorescence intensity of the two fluorescent dyes in the sample sample for fluorescence measurement prepared in the same manner as described above is measured using a single molecule fluorescence analysis system MF20 (Olympus) according to FCCS. The single-molecule fluorescence analysis system calculates the cross-correlation function of two types (two wavelength bands) of fluorescence intensity according to an internal program. When the fluorescence-labeled normal prion is in a brown motion alone in the solution, the cross-correlation function value is substantially 1, but a prion with a different color dye added to one aggregate is incorporated. The correlation function is Large significant value. If the test sample contains abnormal prions, and two types of fluorescently labeled normal prions are incorporated into one aggregate by the conversion reaction, the cross-correlation function is The force is detected that the fluorescent-labeled normal prion is converted to an abnormal form and aggregated. That is, it is detected that an abnormal prion is included in the test sample.

Claims

The scope of the claims

[1] A method for detecting anomalous prions,

A process of preparing a test sample solution by mixing a test sample to be examined for whether or not it contains an abnormal type prion with a normal type prion labeled with fluorescence;

Exposing the test sample solution to reaction conditions in which abnormal prions convert normal prions to abnormal prions;

Measuring the fluorescence intensity of the fluorescent label added to the fluorescent-labeled normal prion in the test sample solution exposed to the reaction conditions;

Based on the fluorescence intensity, the fluorescence-labeled normal prion is present alone in the sample solution! Or is incorporated into the aggregate! The process of determining

When it is determined that the fluorescent-labeled normal prion is incorporated in the aggregate, the test sample contains an abnormal prion that converts the fluorescent-labeled normal prion into an abnormal prion. How to determine that it was.

[2] The method according to claim 1, wherein in the step of preparing the test sample solution, an animal body fluid sample containing normal prion is further mixed.

3. The method according to claim 2, wherein the bodily fluid sample of the animal containing the normal prion is a sample prepared from brain tissue collected from an animal not infected with prion disease.

4. The method according to claim 1, wherein the test sample is an animal tissue or body fluid collected from an animal.

5. The method according to claim 4, wherein the test sample is blood of the animal collected from the animal to be determined whether or not it has an abnormal prion.

6. The method according to claim 1, wherein in the step of measuring the fluorescence intensity, the fluorescence intensity is measured by fluorescence correlation spectroscopy.

7. The method according to claim 1, wherein in the process of measuring the fluorescence intensity, the fluorescence intensity is measured by a fluorescence intensity distribution analysis method.

[8] The method of claim 7, wherein the fluorescent label of each molecule of the fluorescently labeled normal prion A method characterized by the fact that the numbers of knowledge are equal to each other! /.

9. The method according to claim 1, wherein in the process of measuring the fluorescence intensity, the fluorescence intensity is measured by a fluorescence depolarization method.

[10] The method according to claim 1, wherein in the step of preparing the test sample solution, at least two kinds of the fluorescently labeled normal prions mixed with the test sample solution are used. In the process of measuring the fluorescence intensity, the fluorescence intensity is measured by fluorescence cross-correlation spectroscopy, including separate normal prions labeled with fluorescence labels having different fluorescence wavelength characteristics. A method characterized by.

[11] The method of claim 1, wherein the reaction condition is that the test sample solution is at least 30 minutes.

A method that is to incubate at 37 ° C.