JP5841945B2 - Amyloid assay device and amyloid assay method - Google Patents

Description

  The present invention relates to an amyloid assay device and an amyloid assay method.

  Amyloidosis is known as a serious disease in an aging society. Amyloidosis is a general term for diseases in which abnormal protein aggregates called “amyloid fibrils”, which are aggregated with proteins and are not branched with a width of 10 nm, are deposited. Amyloidosis includes about 20 kinds of serious diseases including Alzheimer's disease, type II diabetes, prion disease, dialysis amyloidosis, AL amyloidosis, and Parkinson's disease. Although amyloid fibrils are thought to be a regular assembly of denatured proteins in the axial direction, there are many unclear points in the structure and formation mechanism.

  In recent years, research on amyloidosis has been actively conducted, but it is difficult to predict the deposition of amyloid. The formation of amyloid is a phenomenon similar to the crystal formation of a substance, and even if the causative substance exceeds a dangerous level, the energy barrier for amyloid formation is high, and thus the amyloid formation is often maintained in a supersaturated state. Under such circumstances, amyloid and oligomers thereof are not formed, amyloidosis does not develop, and the patient is in a latent state. A major problem in research on amyloidosis is that it is difficult to predict the formation of associated abnormal aggregates such as amyloid and oligomers. Even in basic research in vitro, the reaction of the causative protein to form amyloid is slow, and in general, amyloid formation requires a long time of several days to several months.

  On the other hand, the inventors of the present application have announced that by irradiating a protein with ultrasonic waves, the energy barrier of amyloid formation is lowered to promote the formation of amyloid fibrils (for example, Non-Patent Documents 1 and 2). reference). Non-Patent Document 1 describes that amyloid fibrils are efficiently induced by ultrasonic treatment. Non-Patent Document 2 describes that fine and homogeneous amyloid particles are formed by antagonizing amyloid fibril formation induction and crushing by ultrasonic treatment.

“Ultrasonication-Induced Amyloid Fibril Formation of Beta 2-Microglobulin”, “Ultrasonication-induced amyloid formation of β2-microglobulin”. Ohashi, Y .; Kihara, M .; Naiki, H .; & Goto, Y .; (2005) J. Org. Biol. Chem. 280, 32843-32848. “Ultrasonication-dependent production and broken-leaded-minimized fibrils”. Ultrasonication-Dependent Production and Breakdown Lead-to-Minimum-sized Amyloid Fibrils. Chatani, E .; Lee, Y .; -H. Yagi, H .; Yoshimura, Y .; Naiki, N .; & Goto, Y .; (2009) Proc. Natl. Acad. Sci. USA, 106, 11119-11124.

  However, even in the methods described in Non-Patent Documents 1 and 2, amyloid assay could not be performed in a short time for each of a plurality of solutions containing proteins.

  The present invention has been made in view of the above problems, and an object thereof is to provide an amyloid assay device and an amyloid assay method capable of performing an amyloid assay for each of a plurality of solutions containing proteins in a short time. It is in.

  An amyloid assay device according to the present invention includes an ultrasonic irradiation unit that irradiates a solution containing a protein placed in each of a plurality of wells of a microplate with an ultrasonic wave, amyloid formed from the protein by the ultrasonic irradiation, and A fluorescence detection unit that detects fluorescence emitted by binding with an amyloid-specific fluorescent dye.

  In one embodiment, the ultrasonic wave irradiation unit irradiates the plurality of wells with ultrasonic waves nonuniformly.

  In one embodiment, the ultrasonic irradiation unit uniformly applies ultrasonic waves to the plurality of wells.

  In one embodiment, the amyloid assay device further includes a sound pressure measurement device that measures the sound pressure of the ultrasonic waves irradiated to each of the plurality of wells of the microplate by the ultrasonic irradiation unit. In a further embodiment, the amyloid specific fluorescent dye comprises thioflavin T.

  In one embodiment, the ultrasonic irradiation unit includes a processing tank, an ultrasonic transducer, and a moving unit that moves the microplate.

  In one embodiment, the moving unit moves the microplate from the ultrasonic wave irradiation unit to the fluorescence detection unit.

  In one embodiment, the moving unit moves the microplate while the ultrasonic wave irradiation unit irradiates the ultrasonic wave to the solution.

  The amyloid assay method according to the present invention includes a step of irradiating a solution containing a protein placed in each of a plurality of wells of a microplate with an ultrasonic wave, and the amyloid formed from the protein and the amyloid-specific fluorescent dye bind to each other. And detecting the fluorescence emitted.

  In one embodiment, in the step of irradiating the ultrasonic waves, the plurality of wells are irradiated with ultrasonic waves non-uniformly.

  In one embodiment, the method further comprises preparing a microplate in which a solution containing a protein and an amyloid-specific fluorescent dye is placed in each of the plurality of wells, and in the step of preparing the microplate, the plurality of wells Put the same solution in each of the.

  In one embodiment, in the step of irradiating with ultrasonic waves, the plurality of wells are irradiated with ultrasonic waves uniformly.

  In one embodiment, the method further comprises preparing a microplate in which a solution containing a protein and an amyloid-specific fluorescent dye is placed in each of the plurality of wells, and in the step of preparing the microplate, the plurality of wells Put a different solution in each.

  In one embodiment, the amyloid assay method further includes a step of moving the microplate to a fluorescence detection unit after detecting the fluorescence and before detecting the fluorescence.

  In one embodiment, the step of irradiating the ultrasonic wave moves the microplate while irradiating the ultrasonic wave to the solution.

  According to the amyloid assay device and the amyloid assay method of the present invention, amyloid assay can be performed in a short time for each of a plurality of solutions containing proteins.

(A) is a schematic diagram of an embodiment of an amyloid assay device according to the present invention, and (b) is a schematic diagram of a microplate used in the amyloid assay device shown in (a). (A) is a schematic diagram of the ultrasonic irradiation apparatus in the amyloid assay apparatus of this embodiment, (b) is a schematic top view of the ultrasonic irradiation apparatus shown in (a), (c) is ( It is typical sectional drawing of the ultrasonic irradiation apparatus shown to a). (A) is a graph which shows the change of the size of the amyloid fiber by irradiation of an ultrasonic wave, (b) is a schematic diagram which shows the atomic force microscope (Atomic Force Microscope: AFM) image when not irradiating an ultrasonic wave. (C) is a schematic diagram which shows an AFM image at the time of irradiating an ultrasonic wave with respect to the amyloid of (b). It is a schematic diagram of the plate reader in the amyloid assay device of this embodiment. It is a schematic diagram of an embodiment of an amyloid assay device according to the present invention. (A) is a schematic diagram of the sound pressure measuring device used for the amyloid assay apparatus of this embodiment, (b) is a schematic diagram of the sensor part front-end | tip of (a). It is a flowchart for demonstrating embodiment of the amyloid assay method by this invention. It is a schematic diagram of the microplate after ultrasonication. It is a graph which shows the time change of the amyloid formation degree depending on ultrasonication. (A)-(d) is a schematic diagram which shows the quantity of the amyloid in case the irradiation time of an ultrasonic wave is 0 minute, 90 minutes, 120 minutes, and 210 minutes, respectively. (A) is a graph which shows the change of the amyloid formation degree according to the difference in protein concentration, (b) is a graph which shows the change of the amyloid formation degree in the physiological conditions similar to a patient internal environment. (A) is a schematic diagram showing the sound pressure distribution of ultrasonic waves in each well, (b) is a schematic diagram showing the degree of amyloid formation, and (c) is a graph showing the relationship between the sound pressure and the amount of amyloid. It is. (A)-(d) is a schematic diagram for demonstrating determination of the risk of the onset of amyloidosis. It is a schematic diagram of the amyloid assay apparatus of this embodiment. (A) is a typical top view of the ultrasonic irradiation apparatus in the amyloid assay apparatus of this embodiment, (b) is a typical side view of (a). It is a graph which shows the time change of the fluorescence intensity from the solution in each well in the plate reader at the time of using the amyloid assay apparatus of this embodiment. It is a schematic diagram which shows the lag time of the solution in each well in the plate reader in the case shown in FIG. (A) is a typical top view of the ultrasonic irradiation apparatus in the amyloid assay apparatus of this embodiment, (b) is a typical side view of (a). (A) is a typical top view of the ultrasonic irradiation apparatus in the amyloid assay apparatus of this embodiment, (b) is a typical side view of (a). It is a schematic diagram of the amyloid assay apparatus of this embodiment. It is a schematic diagram of the plate reader in the amyloid assay apparatus shown in FIG. It is a schematic diagram which shows an example of the plate reader shown in FIG. It is a graph which shows the time change of the fluorescence intensity from the solution in each well in the plate reader at the time of using the amyloid assay apparatus of this embodiment. It is a schematic diagram which shows the lag time of the solution in each well in the plate reader in the case shown in FIG. It is a graph which shows distribution of the lag time according to the presence or absence of the movement of the microplate at the time of ultrasonic irradiation.

  Hereinafter, embodiments of an amyloid assay device and an amyloid assay method according to the present invention will be described with reference to the drawings. However, the present invention is not limited to the following embodiments.

[Amyloid assay device]
An embodiment of an amyloid assay device according to the present invention will be described with reference to the drawings. FIG. 1A shows a schematic diagram of an amyloid assay device 100 of the present embodiment, and FIG. 1B shows a schematic diagram of a microplate P used in the amyloid assay device 100.

  The amyloid assay device 100 includes an ultrasonic irradiation device 10 and a plate reader 20. In the ultrasonic irradiation apparatus 10, a microplate P provided with a plurality of wells (holes or depressions) W is mounted. For example, in the microplate P, 96 wells W are arranged in 12 rows and 8 columns. Each of the plurality of wells W of the microplate P contains a protein solution. The same solution may be put into the plurality of wells W of the microplate P, or different solutions may be put therein. In the present specification, the same solution means that the solution component and the solution concentration are the same, and the different solution means that at least one of the solution component and the solution concentration is different. For example, the same solution includes body fluid (blood, etc.) of the same subject, and the different solution includes body fluid (blood, etc.) of a different subject.

  The ultrasonic irradiation apparatus 10 irradiates the protein solution placed in each well W with ultrasonic waves. When sonication is performed on a supersaturated solution of amyloid-causing protein, amyloid is generated. This is presumably because local high temperature and high pressure are generated, the solution is disturbed, and supersaturation is disturbed. In addition, when an ultrasonic wave is irradiated to an amyloid fiber, the amyloid fiber is fragmented by impact. Although details will be described later, the ultrasonic irradiation apparatus 10 may uniformly irradiate each of the samples placed in the plurality of wells W of the microplate P or may irradiate the ultrasonic waves nonuniformly. Good. In this specification, “irradiating ultrasonic waves to a plurality of samples non-uniformly” means “irradiating at least two samples with ultrasonic waves at different intensities (sound pressures)”, and “ “Uniformly irradiating a plurality of samples” means “irradiating at least two samples with ultrasonic waves at the same intensity (sound pressure)”. Such an ultrasonic irradiation device 10 can promote the formation of amyloid in each of the solutions placed in the plurality of wells W of the microplate P.

  When amyloid is derived from a protein, amyloid emits fluorescence by binding to an amyloid-specific fluorescent dye. The presence of the amyloid-specific fluorescent dye in the plurality of wells W of the microplate P in which amyloid is induced causes fluorescence to be emitted, and the plate reader 20 detects the amyloid in the solution by detecting this fluorescence. Can do.

[Ultrasonic irradiation equipment]
Hereinafter, the ultrasonic irradiation apparatus 10 will be described with reference to FIG. FIG. 2 (a) shows a schematic front view of the ultrasonic irradiation device 10, FIG. 2 (b) shows a schematic top view of the ultrasonic irradiation device 10, and FIG. 2 (c) shows the ultrasonic irradiation device. 10 is a schematic cross-sectional view.

  A microplate P is mounted on the ultrasonic irradiation device 10. The microplate P is preferably formed from a material with good ultrasonic efficiency. For example, the microplate P is formed from polystyrene. Such a microplate P is formed from a mold. In addition, a cap may be attached to the microplate P in case the solution contains an infectious substance. Here, the ultrasonic irradiation apparatus 10 functions as an “ultrasonic irradiation unit that irradiates ultrasonic waves to a solution containing a protein placed in each of a plurality of wells of a microplate”.

  The ultrasonic irradiation apparatus 10 includes a processing tank 12 and an ultrasonic transducer 14. The treatment tank 12 has side plates 12a and 12b facing each other bent inward by 45 degrees at the center, side plates 12c and 12d facing each other that communicate with the side plates 12a and 12b, and a bottom plate 12e connected to the side plates 12a to 12d. ing. The treatment tank 12 is filled with water. The ultrasonic waves generated in the ultrasonic transducer 14 are transmitted through water. The ultrasonic transducers 14 are attached vertically to the inclined surfaces 12a2 and 12b2 of the side plates 12a and 12b, respectively. In addition, here, a diaphragm 14a to which the ultrasonic transducer 14 is attached is disposed inside the inclined surfaces 12a2 and 12b2, and the ultrasonic waves intersect each other at a right angle so that the energy becomes stronger, and amyloid An effective crushing operation of the fiber is performed. In addition, a microplate P is placed on top of the processing tank 12. The microplate P is fixed by a holder (not shown) with the tip of each well W immersed in water. This holder is arranged at the position where the ultrasonic wave is most easily received in the central portion of the treatment tank 12.

  When the ultrasonic vibrator 14 vibrates and oscillates, the ultrasonic wave passes through the vibration plate 14a and becomes strong and travels toward each well W of the microplate P. At this time, since the ultrasonic waves travel in a direction perpendicular to the respective inclined plates 14a, the ultrasonic waves intersect at right angles. Then, this intersection is set in advance near the central water surface of the treatment tank 12, and each well W of the microplate P is arranged in the vicinity thereof. The ultrasonic wave passes through the water in the treatment tank 12 and reaches each well W of the microplate P, and further reaches the solution in each well W of the microplate P to induce and crush amyloid formation.

  In addition, although the ultrasonic transducer | vibrator 14 and the inclination board 14a are arrange | positioned at two places of the side plates 12a and 12b in the ultrasonic irradiation apparatus 10, the ultrasonic transducer 14 and the inclination plate 14a are not only the side plates 12a and 12b but also the side plate. You may attach to 12c, 12d. In addition, when more powerful crushing is required, the ultrasonic transducer 14 and the inclined plate 14a may be attached to the bottom plate 12e.

  Here, with reference to FIG. 3, the difference in the size of amyloid fibrils due to the irradiation of ultrasonic waves will be described. FIG. 3 (a) schematically shows the relationship between the size of amyloid fibrils and free energy. FIG. 3B shows an AFM image of amyloid when no ultrasonic wave is irradiated, and FIG. 3C shows an atomic force microscope image of amyloid when the ultrasonic wave is irradiated.

  When a supersaturated protein is allowed to stand for a long time (several days to several months), since the energy barrier for nucleation is large, a reaction exceeding the energy barrier occurs with a low probability, and amyloid fibrils are generated. For this reason, the formation of amyloid fibrils has a long incubation period of several days to several months, and the reproducibility of the experiment is low. On the other hand, when an ultrasonic wave is irradiated, the energy barrier for nucleation can be greatly reduced with good reproducibility. In addition, when irradiating ultrasonic waves, amyloid fibers are elongated, but large-sized fibers are easily crushed. Therefore, amyloid fibrils having a relatively uniform size can be formed. Thus, the irradiation with ultrasonic waves can promote the formation of amyloid and improve the reproducibility.

[Plate reader]
FIG. 4 shows a schematic diagram of the plate reader 20. The plate reader 20 is also called a microplate reader. Here, the plate reader 20 functions as a “fluorescence detection unit that detects fluorescence emitted by the binding of amyloid formed from protein by irradiating ultrasonic waves and an amyloid-specific fluorescent dye”.

  A microplate P is mounted on the plate reader 20 and fixed. Here, the microplate P is preferably the same as that mounted in the ultrasonic irradiation apparatus 10. When the cap is attached to the microplate P, it is preferable that the cap has high transparency. However, the plate reader 20 may be mounted with a microplate P different from that mounted in the ultrasonic irradiation apparatus 10.

  The plate reader 20 includes a light source 22 and a light receiving unit 24. For example, the light receiving unit 24 is a photomultiplier tube. Of the light emitted from the light source 22, light having a specific wavelength selected by the grating is irradiated to the well W of the microplate P via the optical fiber fb1. Here, spectroscopy is performed twice. Light irradiation and light reception are performed on each well W of the microplate P. For this purpose, the optical fibers fb1 and fb2 are configured to be operable in a two-dimensional direction with respect to the main surface of the microplate P to which the tips are fixed. Here, the optical fibers fb1 and fb2 are moved while the microplate P is fixed to perform light irradiation and fluorescence detection. However, the microplate P is moved without moving the light emitted toward the microplate P. It may be moved.

  Fluorescence is generated from the solution in the well W by light irradiation. The generated fluorescence is transmitted to the light receiving unit 24 via the optical fiber fb2, and the intensity thereof is measured. By moving the optical fibers fb1 and fb2 corresponding to the wells W of the microplate P, the plate reader 20 can evaluate many samples. In addition, when using the amyloid assay apparatus 100 for the basic research field of amyloid, it is preferable that the plate reader 20 selects a wavelength arbitrarily using a grating element.

  When the microplate P used for the ultrasonic irradiation apparatus 10 and the plate reader 20 is the same, the observer may move the microplate P. Or the ultrasonic irradiation apparatus 10 and the plate reader 20 may be comprised integrally.

[Integrated amyloid assay device]
FIG. 5 shows an integrated amyloid assay device 100 ′ in which the ultrasonic irradiation device 10 and the plate reader 20 are integrated. In the amyloid assay device 100 ′, the microplate P is provided with optical fibers fb 1 and fb 2 in the upper part of the processing tank 12.

[Sound measuring device]
The amyloid assay device 100 and the integrated amyloid assay device 100 ′ further include a sound pressure measurement device that measures the sound pressure of the ultrasonic waves irradiated to the plurality of wells W of the microplate P by the ultrasonic irradiation device 10. Also good. The sound pressure measuring device measures the sound pressure of the ultrasonic wave irradiated to each well W of the microplate P.

  FIG. 6A shows the sound wave measuring device 30. The sound pressure measuring device 30 includes a sensor unit 32, a voltage monitor unit 34, and a processing unit 36 that processes values monitored by the voltage monitor unit 34. At the time of sound pressure measurement, sound pressure is measured in a state where the sensor unit 32 is infiltrated into the solution in the well W of the microplate P. The sound pressure measurement and the fluorescence measurement may be performed separately or simultaneously.

  The sensor unit 32 converts the sound pressure of the ultrasonic waves into a potential, and the voltage monitor unit 34 displays the potential waveform converted in the sensor unit 32. In the processing unit 36, the sound pressure, frequency, and harmonic component of the ultrasonic wave can be obtained. The processing unit 36 may perform filtering or fast Fourier transform (FFT).

  FIG. 6B shows an example of the tip of the sensor unit 32. The sensor unit 32 includes a rod-shaped main body 32a and a piezoelectric element 32b provided at one end of the main body 32a. The main body 32a is insulated at least on the surface. The main body 32a is an alumina lot having a diameter of about 4 mm and a length of about 100 mm, for example. The piezoelectric element 32b has a pair of metal electrodes 32b1 and 32b2 and a piezoelectric body 32b3 sandwiched between them. Each of the metal electrodes 32b1 and 32b2 has a laminated structure of chromium and gold and has a thickness of 500 nm. The piezoelectric body 32b3 is made of, for example, lead zirconate titanate (PZT), polyvinylidene fluoride (PVDF), quartz or langasite (La3Ga5SiO14). In addition, it is preferable that the surface including the front-end | tip of the sensor part 32 is protected by the protective layer 32c. The protective layer 32c is formed from, for example, an epoxy resin.

[Amyloid assay method]
Hereinafter, an embodiment of the amyloid assay method according to the present invention will be described with reference to FIG.

  As shown in S22, a microplate P in which a protein solution is placed in each of the plurality of wells W is prepared. Preferably, the solution also contains an amyloid specific fluorescent dye. For example, the amyloid specific fluorescent dye is Thioflavin T.

  Next, as shown in S <b> 24, the solution placed in each of the plurality of wells W of the microplate P is irradiated with ultrasonic waves. As described above, the sound pressure of the ultrasonic wave applied to each solution of the plurality of wells W may be uniform or non-uniform.

  Next, as shown in S26, the fluorescence emitted by the binding of the amyloid derived from the protein and the amyloid-specific fluorescent dye is detected. The fluorescence is detected by the plate reader 20. Finally, the protein (amyloid) and the solution are discarded.

  The amyloid assay device 100 and the integrated amyloid assay device 100 'can be used to study abnormal protein aggregation. Alternatively, the amyloid assay device 100 and the integrated amyloid assay device 100 'are used to determine the risk of developing amyloidosis.

  The embodiment of the amyloid assay device and the amyloid assay method of the present invention has been described above with reference to FIGS.

  Hereinafter, application examples of the amyloid assay devices 100 and 100 ′ will be described with reference to FIGS. 8 to 13.

  First, the amyloid of β2 microglobulin that causes dialysis amyloidosis will be specifically described. Currently, there are over 260,000 long-term hemodialysis patients in the country. Many patients who undergo long-term hemodialysis for 10 years or more develop “carpal tunnel syndrome” that causes severe pain and deformation of the bone and joint, and this is caused by amyloid deposition formed by β2 microglobulin. β2 microglobulin is essentially a component of a histocompatibility antigen that is responsible for immune functions and an essential protein that is responsible for vital functions. In dialysis patients, β2 microglobulin is not degraded and the protein concentration in the blood increases. If this condition persists for many years, it is thought to cause amyloid deposition, but the detailed pathogenesis is unknown.

  For example, the formation of β2 microglobulin amyloid fibrils generally takes several days to one month when the test tube is left standing. On the other hand, in the amyloid assay device 100, even if the concentration of β2 microglobulin is low (for example, in vivo concentration: 0.05 mg / ml), formation of amyloid of β2 microglobulin is performed using ultrasound. In addition to facilitating (on the order of 20 minutes), the amyloid can be assayed in a short time (eg, hundreds of samples within minutes).

  When β2 microglobulin amyloid is formed by the amyloid assay device 100, the sound pressure provided to the solution in each well W of the microplate P is, for example, 0 to 0.5 MPa, and the value of the sound pressure is further adjusted. can do. Here, for example, the sound pressure (average value ± standard deviation) provided to the solution in each well W of the microplate P is 34600 ± 17000 Pa, and the frequency of the ultrasonic vibrator by the ultrasonic irradiation device 10 is 17. The intensity of the ultrasonic transducer is 350 or 700 Watt. Note that the sound pressure provided to the solution in each well W, the vibration frequency of the ultrasonic vibrator by the ultrasonic irradiation device 10, and the strength of the ultrasonic vibrator are not limited to the above values. In the case where “amyloid is formed from protein by ultrasonic irradiation to a protein-containing solution”, the sound pressure provided to the solution in each well W, the frequency of the ultrasonic vibrator by the ultrasonic irradiation device 10 The value of the intensity of the ultrasonic transducer is arbitrary. The amyloid-specific fluorescent dye is, for example, thioflavin T. In this case, when the plate reader 20 irradiates light having a wavelength of around 450 nm as excitation light, the plate reader 20 can detect fluorescence around 490 nm emitted from amyloid.

  FIG. 8 shows a schematic diagram of the microplate P after the ultrasonic treatment. Here, an acidic condition of pH 2 and a protein concentration of 0.3 mg / ml were repeatedly subjected to ultrasonic irradiation for 1 minute and interruption for 9 minutes. In the well W near the center of the microplate P, amyloid formation proceeded in the same manner.

  FIG. 9 shows the change over time in the degree of amyloid formation. Here, in order to investigate the influence of ultrasonic irradiation, group A which repeated ultrasonic irradiation for 1 minute and interruption for 9 minutes was compared with group B which repeated ultrasonic irradiation for 10 minutes and interruption for 5 minutes. did. In the case of repeating ultrasonic irradiation for 1 minute and interruption for 9 minutes, amyloid formation starts in about 90 minutes, whereas in the case of repeated irradiation with ultrasonic waves for 10 minutes and interruption for 5 minutes, it takes about 20 minutes. Amyloid formation begins. Thus, amyloid can be more efficiently formed by arbitrarily changing the ultrasonic treatment time. The plurality of curves in each of the groups A and B show data of a plurality of wells having different sound pressures of ultrasonic waves irradiated at the same time. One measurement shows that the experiment is highly reproducible. For comparison, the microplate P was allowed to stand without performing ultrasonic irradiation, but in this case, amyloid was not formed.

  FIG. 10A to FIG. 10D are schematic diagrams showing the amount of amyloid when the ultrasonic irradiation time is 0 minutes, 90 minutes, 120 minutes, and 210 minutes, respectively. It is understood that the number of wells in which amyloid is formed increases with the irradiation time and the amount of amyloid increases in each well.

  Here, with reference to FIG. 11, a change in the degree of amyloid formation in which the maximum value is normalized to 1 when the amyloid assay device 100 or the integrated amyloid assay device 100 'is used will be described. FIG. 11 (a) shows the change in the degree of amyloid formation according to the difference in protein concentration. Here, the solution is acidified, and ultrasonic irradiation for 1 minute and interruption for 9 minutes are repeated. Here, the protein concentration is 0.3 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml. In any case, when the irradiation time increases to a certain extent, the degree of amyloid formation increases. However, the higher the protein concentration, the shorter the time at which the degree of amyloid formation starts to increase. The protein concentration in the patient is approximately 0.05 mg / ml. By using the amyloid assay device 100, amyloid formation can be detected even at such a low concentration.

  FIG. 11B shows changes in the degree of amyloid formation under physiological conditions similar to those in the patient internal environment. The two graphs show data of two wells having a similar sound pressure level of the irradiated ultrasonic wave among the plurality of wells of the microplate P. Here, the protein concentration is 0.05 mg / ml and the solution is almost neutral. As understood from the comparison with FIG. 11 (a), although it took some time, it was confirmed that amyloid was formed even in this case. In addition, as shown in FIG. 11B, the reproducibility of the reaction can be observed and evaluated by using a plurality of wells W of the microplate P at the same time.

  The amyloid assay device 100 and the integrated amyloid assay device 100 'can be suitably applied to the prevention and diagnosis of amyloidosis. Specifically, the risk level of developing amyloidosis can be determined using the amyloid assay device 100 or the integrated amyloid assay device 100 ′. As described above, the ultrasonic irradiation apparatus 10 can provide a non-uniform or uniform sound pressure to the solution in the well W. For example, whether the sound pressure applied to the solution in the well W is nonuniform or uniform can be switched by an irradiation state switching unit provided in the ultrasonic irradiation apparatus 10. When the ultrasonic irradiation apparatus 10 provides a non-uniform sound pressure to the solution of the well W, it is preferable to put the same sample (for example, blood of the same subject) into each well W of the microplate P. Or when the ultrasonic irradiation apparatus 10 provides a uniform sound pressure to the solution of the well W, it is preferable to put different samples (for example, blood of different subjects) in each well W of the microplate P.

  First, the case where the ultrasonic irradiation apparatus 10 provides a non-uniform sound pressure to the solution of the well W of the microplate P is demonstrated. The sound pressure can be measured by the sound pressure measuring device 30 described above with reference to FIG. FIG. 12A is a schematic diagram showing the sound pressure distribution of ultrasonic waves in each well, and FIG. 12B is a schematic diagram showing the degree of amyloid formation. 12A and 12B, the vertical axes A to H correspond to the first to eighth rows of the microplate P, and the horizontal axes 1 to 12 represent the first of the microplate P. It corresponds to the 12th line. Here, each well W of the microplate P contains the same sample (for example, blood of the same subject). As understood from comparison with FIG. 12 (a) and FIG. 12 (b), it is understood that the amyloid in the well with higher sound pressure has a higher degree of formation. FIG. 12C shows the relationship between the sound pressure and the amount of amyloid. Thus, the sound pressure and the amount of amyloid have a substantially proportional relationship. Although each subject has a different risk of developing amyloidosis, the risk level of developing amyloidosis can be specified by specifying the sound pressure at which a predetermined amount of amyloid is formed during a predetermined irradiation time.

  In addition, when the ultrasonic irradiation apparatus 10 provides a uniform sound pressure to the solution in the well W, it is preferable that a different sample is placed in each well W of the microplate P. Specifically, blood or the like of different subjects is put in each well W of the microplate P.

  Here, the determination of the risk of developing amyloidosis in different subjects will be described with reference to FIG. FIG. 13 (a) shows different subjects. Blood or the like is collected from different subjects, and the solution is put into different wells W of the microplate P as shown in FIG. Thereafter, when each well W is irradiated with ultrasonic waves of uniform sound pressure, a predetermined amount of amyloid is formed in the solution of the subject having a relatively high risk of developing amyloidosis, as shown in FIG. A predetermined amount of amyloid is not formed in the solution of a subject with a relatively low risk of developing amyloidosis. For this reason, by setting the ultrasonic wave to be a threshold for the risk of developing amyloidosis, it is possible to determine whether the subject is positive or negative as shown in FIG.

  In the integrated amyloid assay device 100 ′ described above with reference to FIG. 5, the plate reader 20 measures the fluorescence emitted from the solution while the microplate P is in the treatment tank 12. However, the plate reader 20 may measure fluorescence emitted from the solution of the microplate P that has moved to the outside of the processing tank 12.

  FIG. 14 shows a schematic diagram of an integrated amyloid assay device 100 ′. The integrated amyloid assay device 100 ′ includes an ultrasonic irradiation device 10 and a plate reader 20. For example, MTP-810 or SH-9000 manufactured by Corona Electric Co., Ltd. is used as the plate reader 20. A microplate having 96 wells W is used as the microplate P. For example, the microplate P may have 96 wells W of a half area black type. In addition, after putting a solution in the well W of the microplate P, in order to prevent evaporation etc., it is preferable to seal the well W. Moreover, when sealing, it is preferable to use a thing with little autofluorescence as a sealing member.

  The ultrasonic irradiation apparatus 10 includes a processing tank 12, an ultrasonic transducer 14, and a moving unit 16 that moves the microplate P in the processing tank 12. For example, water is stored in the treatment tank 12. Also here, the vibration plate 14 a is provided inside the treatment tank 12. For example, after the ultrasonic irradiation apparatus 10 irradiates the ultrasonic wave, the moving unit 16 moves the microplate P from the inside of the processing tank 12 to the outside of the processing tank 12, and then the plate reader 20 moves the microplate P of the microplate P. Fluorescence emitted from the solution in the well W is detected.

  Hereinafter, an example of the ultrasonic irradiation apparatus 10 will be described with reference to FIG. The moving part 16 includes a support part 16t, an elevating part 16u attached to the support part 16t, and a fixed base 16v on which the microplate P is placed. The fixed base 16v is attached to the elevating part 16u, and the elevating part 16u moves the fixed base 16v in the z direction. Accordingly, the microplate P moves from the inside of the processing tank 12 to the outside of the processing tank 12 or from the outside of the processing tank 12 into the processing tank 12.

  The microplate P is moved from the inside of the processing tank 12 to the plate reader 20 (FIG. 14) outside the processing tank 12 by the elevating unit 16 u, and then the plate reader 20 is amyloid in the solution of the well W in the microplate P. Is detected. As described above, when amyloid fibrils are induced, amyloid emits fluorescence by binding to an amyloid-specific fluorescent dye, and amyloid can be detected by measuring this fluorescence. Here, when irradiating with ultrasonic waves, the microplate P remains fixed.

  For example, β2 microglobulin is used as the protein, and thioflavin T is used as the amyloid-specific fluorescent dye. 200 μL of 0.3 mg / ml β2 microglobulin solution (containing 100 mM NaCl, 5 μM thioflavin T, pH 2.5) is placed in a microplate P having 96 wells W of the half area black type. For example, the microplate P is set to a temperature of 37 ° C. The ultrasonic irradiation apparatus 10 repeats ultrasonic irradiation for 1 minute and interruption for 9 minutes.

  FIG. 16 shows a temporal change in fluorescence intensity when ultrasonic waves are irradiated. Here, attention is focused on the time from the start of the irradiation of ultrasonic waves to the start of the increase in fluorescence intensity. The shortest time from the start of ultrasonic irradiation to the start of the increase in fluorescence intensity is about 50 minutes, and the fluorescence intensity from the solution in most wells starts increasing within 180 minutes. In the following description of the present specification, the time from the start of ultrasonic irradiation to the start of increase in fluorescence intensity may be referred to as lag time.

  In FIG. 17, the schematic diagram of the microplate P showing lag time is shown. In FIG. 17, the wells in the first row to the twelfth row are indicated as 1, 2, 3,... 12 respectively, and the wells in the first column to the eighth column are indicated as A, B,. As understood from FIG. 17, the lag time of the solution in most wells W near the center in the microplate P is about 100 minutes, and the lag time of the solution in the wells W near the end is about 120 minutes. is there. The lag time of the solution in the well W at the end of the microplate P is longer than the lag time of the solution in the well W near the center.

  In the ultrasonic irradiation apparatus 10 shown in FIG. 15, after the ultrasonic wave irradiation is performed in the processing tank 12, the moving unit 16 moves the microplate P to the plate reader 20. It is not limited to. While the ultrasonic irradiation is performed, the moving unit 16 may move the microplate P. For example, the moving unit 16 may rotate the microplate P around a predetermined point, and thereby the microplate P may be rotated. In this case, the rotation speed of the microplate P is preferably in the range of 0.5 rpm to 11 rpm, and the rotation speed of the microplate P is more preferably about 6 rpm. Alternatively, the microplate P may move around by the moving unit 16 or may move in parallel. As described above, the moving unit 16 moves the microplate P in the processing tank 12, and thereby can irradiate the solution in each well W of the microplate P almost uniformly with ultrasonic waves.

  Hereinafter, an example of the ultrasonic irradiation apparatus 10 will be described with reference to FIG. The moving part 16 is attached so as to be movable along the slide part 16s1, a support part 16t1 attached so as to be movable along the slide part 16s1, a slide part 16s2 fixed to the support part 16t1, and a slide part 16s2. And a supporting portion 16t2. The microplate P is placed on the tip of the support portion 16t2. In the following description of the present specification, the slide portions 16s1 and 16s2 are referred to as the first slide portion 16s1 and the second slide portion 16s2, and the support portions 16t1 and 16t2 are referred to as the first support portion 16t1 and the second support portion 16t2. is there.

  The support portion 16t1 moves in the y direction along the slide portion 16s1, and accordingly, the microplate P also moves in the processing tank 12 in the y direction. Further, the support portion 16t2 moves in the x direction along the slide portion 16s2, and accordingly, the microplate P also moves in the processing tank 12 in the x direction. Thus, the moving unit 16 can move the microplate P in the processing tank 12 in the plane direction.

  In the ultrasonic irradiation apparatus 10 described with reference to FIG. 18, the moving unit 16 can move the microplate P along the x direction and the y direction, but the present invention is not limited to this. The moving unit 16 may be capable of moving the microplate P along only one of the x direction and the y direction. For example, the first slide portion 16s1 and the first support portion 16t1 may be omitted from the ultrasonic irradiation apparatus 10 described with reference to FIG. Or the 2nd slide part 16s2 and the 2nd support part 16t2 may be abbreviate | omitted from the ultrasonic irradiation apparatus 10 demonstrated with reference to FIG.

  Moreover, in the ultrasonic irradiation apparatus 10 demonstrated with reference to FIG. 18, the moving part 16 moved the microplate P in the plane direction in the processing tank 12, However, This invention is not limited to this. The moving unit 16 may move the microplate P also in the vertical direction.

  Hereinafter, an example of the ultrasonic irradiation apparatus 10 will be described with reference to FIG. In the ultrasonic irradiation apparatus 10 shown in FIG. 19, the moving unit 16 has the configuration shown in FIG. 18 except that the moving unit 16 further includes an elevating unit 16 u attached to the support unit 16 t 2 and a fixing base 16 v on which the microplate P is placed. It has the same configuration as that of the moving unit 16 described above with reference to the description, and redundant description is omitted to avoid redundancy.

  The fixed base 16v is attached to the elevating part 16u, and the elevating part 16u moves the fixed base 16v in the z direction. Accordingly, the microplate P moves from the inside of the processing tank 12 to the outside of the processing tank 12 or from the outside of the processing tank 12 into the processing tank 12. Although detailed description is omitted here, the microplate P can be moved in three directions orthogonal to each other by the slide parts 16s1, 16s2 and the elevating part 16u. In the ultrasonic irradiation apparatus 10 shown in FIG. 19, the moving unit 16 moves the microplate P when irradiating ultrasonic waves, and can move the microplate P to the plate reader 20 after the ultrasonic irradiation. it can.

  Here, an example of the integrated amyloid assay device will be described with reference to FIG. The integrated amyloid assay device 100 ′ includes an ultrasonic irradiation device 10 that irradiates a solution in the well W of the microplate P with ultrasonic waves, and a plate that detects fluorescence emitted from the amyloid in the solution of the well W in the microplate P. And a reader 20. In addition to the processing tank 12 and the ultrasonic vibrator 14, the ultrasonic irradiation apparatus 10 includes a circulation tank 12a used for circulating the liquid in the processing tank 12, a control unit 14s that controls the ultrasonic vibrator 14, And a drive unit 14t for driving the sound wave vibrator 14. In order to further improve the uniformity of ultrasonic irradiation to the solution in each well W, the number of ultrasonic transducers 14 is preferably as large as possible.

  When a sensor is attached to the processing tank 12 to which the ultrasonic transducer 14 is attached in order to measure the water temperature, the sensor may be damaged due to sound waves. For this reason, it is preferable to attach the sensor which measures water temperature to the circulation tank 12a, and to measure the temperature of the circulation tank 12a.

  FIG. 21 shows a schematic diagram of the plate reader 20 in the amyloid assay device 100 or the integrated amyloid assay device 100 ′ of the present embodiment. The plate reader 20 includes a light source 22, an input optical system Oa, an output optical system Ob, and a light receiving unit 24. As the light receiving unit 24, for example, a photomultiplier tube is used. The light emitted from the light source 22 is applied to the solution in the well W of the microplate P through the input optical system Oa. When fluorescence is emitted from the solution, the fluorescence is detected by the light receiving unit 24 via the output optical system Ob.

  Hereinafter, an example of the input optical system Oa and the output optical system Ob will be described with reference to FIG. FIG. 22 shows a schematic diagram of the plate reader 20. The input optical system Oa includes lenses L1 to L4, slits S1 to S3, a color filter C1, gratings G1 and G2, and a half mirror H1. The output optical system Ob includes slits S4 to S6, a color filter C2, gratings G3 and G4, and a half mirror H2.

  For example, the light emitted from the light source 22 is reflected by the grating G1 through the lenses L1, L2, the color filter C1, and the slit S1. For example, the f value of the lenses L1 and L2 is 60. Also. The color filter C1 blocks light having a wavelength other than the specific wavelength. The reflected light is reflected by the grating G2 through the slit S2. Thereafter, the light passes through the slit S3, the half mirror H1, and the lenses L3 and L4, and finally irradiates the solution in each well of the microplate P via the half mirror H2. Note that the intensity of the light whose traveling direction is changed by the half mirror H1 may be detected by the photodetector PD. Based on this intensity, the intensity of light irradiated to the solution in each well of the microplate P can be easily adjusted.

  When fluorescence is emitted from the solution in each well W of the microplate P, the direction of travel of the fluorescence is bent through the half mirror H2, and reflected by the grating G3 through the slit S4. The reflected light is reflected by the grating G4 through the slit S5. Thereafter, the light finally enters the light receiving unit 24 through the color filter C2 and the slit S6.

  Hereinafter, the induction of amyloid fibrils when the microplate P is moved during ultrasonic irradiation will be described with reference to FIGS. By moving the microplate P during ultrasonic irradiation, the lag time can be shortened and variations in the lag time can be suppressed.

  Again, β2 microglobulin is used as the protein, and thioflavin T is used as the amyloid-specific fluorescent dye. 200 μL of 0.3 mg / ml β2 microglobulin solution (containing 100 mM NaCl, 5 μM thioflavin T, pH 2.5) is placed in a microplate P having 96 wells W of the half area black type. For example, the microplate P is set to a temperature of 37 ° C.

  The moving unit 16 moves the microplate P when irradiating ultrasonic waves. Here, the moving unit 16 rotates the microplate P. The rotation speed of the microplate P is about 6 rpm. The ultrasonic irradiation apparatus 10 repeats ultrasonic irradiation for 1 minute and interruption for 9 minutes.

  FIG. 23 shows temporal changes in fluorescence intensity when the ultrasonic wave is irradiated while moving the microplate P. FIG. Here, focusing on the lag time, the shortest lag time is about 50 minutes, and the longest is about 100 minutes.

  FIG. 24 is a schematic diagram of a microplate showing the lag time when irradiating ultrasonic waves while moving the microplate. In FIG. 24, the wells in the first row to the twelfth row are indicated as 1, 2, 3,... 12 respectively, and the wells in the first column to the eighth column are indicated as A, B,. As understood from FIG. 24, the lag time of the solution in most wells is about 70 to 80 minutes.

  16 and 17 described above show the change in fluorescence intensity with time and the lag time of the MyProplate P when the microplate P is fixed and similarly irradiated with ultrasonic waves. As can be understood from the comparison between FIG. 16 and FIG. 17 and FIG. 23 and FIG. 24, by irradiating the ultrasonic wave while moving the microplate P, the average of the lag time is shortened and the variation of the lag time is suppressed. can do.

FIG. 25 shows the lag time distribution. FIG. 25 shows the distribution according to the presence or absence of the movement of the microplate. The number of samples is 96. Table 1 shows the average lag time and standard deviation according to the presence or absence of movement.

  From FIG. 25 and Table 1, it is understood that the average time of the lag time can be shortened and the variation can be suppressed by moving the microplate during the ultrasonic irradiation.

  In the above description, β2 microglobulin causing dialysis amyloidosis is exemplified as a protein, but the present invention is not limited to this. Other proteins may be used. For example, using amyloid β peptide that causes Alzheimer's disease, IAPP (Isle amyloid polypeptide) that causes type II diabetes, antibody L chain that causes AL amyloidosis, and α-synuclein that causes Parkinson's disease Also good. In the amyloid assay device 100 and the integrated amyloid assay device 100 ′, the formation of amyloid from the protein is promoted by ultrasonic waves, and the formed amyloid is detected.

  For example, amyloid fibrils formed by a peptide called IAPP are responsible for type II diabetes. In type II diabetes, the number of insulin receptors decreases or the function thereof decreases. If such a state continues, the islets of Langerhans β cells that synthesize insulin in the pancreas will also decrease and die. As a cause of β-cell death, the theory that “IAPP synthesized in the pancreas together with insulin forms amyloid fibrils and deposits on the islets of Langerhans, thereby killing β-cells” is prominent.

  Alzheimer's disease progresses by the degeneration and death of neurons that make up the brain. Originally, amyloid formation of amyloid β peptide called senile plaque occurred in the patient's brain, so it was thought that amyloid formation was the direct cause, but recently it is soluble and relatively small called oligomer Abnormal aggregates have been shown to have strong cytotoxicity, which is considered a direct cause of neuronal cell death. By using the ultrasonic irradiation apparatus 10, an oligomer can be assayed by performing ELISA (Enzyme-Linked Immunosorbent Assay) using this oligomer and a specific antibody.

  In the above description, the ultrasonic irradiation apparatus 10 and the plate reader 20 are used for the amyloid assay, but the present invention is not limited to this. The object of formation and assay by the ultrasonic irradiation apparatus 10 and the plate reader 20 is not limited to amyloid, and may be any expression that is expressed by the ultrasonic irradiation apparatus 10 and can be read by the plate reader 20.

  According to the present invention, an amyloid assay can be performed in a short time for each of a plurality of solutions containing a protein. The amyloid assay according to the invention is widely used not only in basic research but also in the areas of prevention and clinical medicine.

DESCRIPTION OF SYMBOLS 10 Ultrasonic irradiation apparatus 20 Plate reader 30 Sound pressure measuring apparatus 100 Amyloid assay apparatus 100 'integrated amyloid assay apparatus

Claims (13)

An ultrasonic irradiation unit that irradiates a solution containing protein put in each of a plurality of wells of a microplate with ultrasonic waves;
A fluorescence detection unit that detects fluorescence emitted by the binding of amyloid formed from the protein and amyloid-specific fluorescent dye by irradiation of the ultrasonic wave,
The ultrasonic irradiation unit has a processing tank and a moving unit,
The moving unit is
While the ultrasonic wave irradiation unit is irradiating the ultrasonic wave to the solution, the microplate is moved in the processing tank,
An amyloid assay device that moves the microplate to the fluorescence detection unit by moving the microplate from the inside of the processing tank to the outside of the processing tank after the irradiation of the ultrasonic waves.   The amyloid assay device according to claim 1, wherein the ultrasonic irradiation unit irradiates the plurality of wells with ultrasonic waves nonuniformly.   The amyloid assay device according to claim 1, wherein the ultrasonic irradiation unit uniformly applies ultrasonic waves to the plurality of wells.   4. The sound pressure measurement device according to claim 1, further comprising a sound pressure measurement device that measures sound pressures of ultrasonic waves irradiated to each of the plurality of wells of the microplate by the ultrasonic wave irradiation unit. 5. Amyloid assay device.   The amyloid assay apparatus according to claim 1, wherein the amyloid-specific fluorescent dye includes thioflavin T. The amyloid assay device according to claim 1, wherein the ultrasonic irradiation unit further includes an ultrasonic transducer.   7. The amyloid assay device according to claim 1, wherein the ultrasonic irradiation unit repeats irradiation of the ultrasonic wave for a first predetermined time and interruption of a second predetermined time. 8. Irradiating a solution containing protein placed in each of a plurality of wells of a microplate with ultrasound;
A step of detecting fluorescence emitted by the binding of the amyloid formed from the protein and the amyloid-specific fluorescent dye with a fluorescence detector;
A step of moving the microplate to the fluorescence detection unit by moving the microplate from the inside of the processing tank to the outside of the processing tank by the moving unit after the ultrasonic wave irradiation and before detecting the fluorescence. It encompasses the door,
The step of irradiating the ultrasonic wave is an amyloid assay method in which the microplate is moved in the processing tank while the ultrasonic wave is being applied to the solution .   The amyloid assay method according to claim 8, wherein in the step of irradiating ultrasonic waves, the plurality of wells are irradiated with ultrasonic waves nonuniformly. Providing a microplate in which a solution containing a protein and an amyloid-specific fluorescent dye is placed in each of the plurality of wells;
The amyloid assay method according to claim 9, wherein in the step of preparing the microplate, the same solution is placed in each of the plurality of wells.   The amyloid assay method according to claim 8, wherein in the step of irradiating with ultrasonic waves, the plurality of wells are irradiated with ultrasonic waves uniformly. Providing a microplate in which a solution containing a protein and an amyloid-specific fluorescent dye is placed in each of the plurality of wells;
The amyloid assay method according to claim 11, wherein in the step of preparing the microplate, different solutions are placed in each of the plurality of wells.   13. The amyloid assay method according to claim 8, wherein the step of irradiating the ultrasonic wave repeats the irradiation of the ultrasonic wave for a first predetermined time and the interruption of the second predetermined time.