Amyloid fibril formation method, amyloid fibril formation apparatus, and amyloid fibril analysis system

Abstract

To provide a new method for forming amyloid fibrils, by which the amyloid fibrils can simply be formed compared with a conventional method.SOLUTION: A method of forming amyloid fibrils from an amyloid fibril forming protein, the method comprising: flowing a fluid comprising the amyloid fibril forming protein into a fluid tube; and conveying the fluid by means for moving the fluid by applying pressure and vacuum to the fluid through the fluid tube by progressively compressing the fluid tube in one direction, thereby forming the amyloid fibrils from the amyloid fibril forming protein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a novel method for forming amyloid fibrils, an apparatus for forming amyloid fibrils, and an amyloid fibril analysis system. [Background technology]

[0002] Amyloid fibrils are rigid fibril-like deposits of amyloid-causing proteins (hereinafter referred to as "amyloid fibril-forming proteins" or simply "amyloid fibril-forming proteins") with a width of 10 nm and a length of several micrometers. They are the causative agents of various amyloidoses, including Alzheimer's disease, Parkinson's disease, prion disease, and dialysis-related amyloidosis. The causative proteins specific to each amyloidosis undergo a supersaturated state to form amyloid fibrils. Amyloid fibrils differ from single solute crystals. However, just as single crystals must overcome an energy barrier to form from supersaturated solutes, amyloid fibrils must overcome a high energy barrier to form from supersaturated causative proteins. Furthermore, just as the presence of seed crystals lowers the energy barrier for crystal formation, the presence of "seed amyloid" promotes the formation of amyloid fibrils.

[0003] Ultrasound irradiation is an effective method for lowering the energy barrier for amyloid fibril formation and inducing the formation of amyloid fibrils from a supersaturated solution of amyloid-causing proteins. Amyloid fibrils can be detected using the fluorescent dye thioflavin T, which is an amyloid-specific fluorescent dye. By combining ultrasound irradiation with thioflavin T and a plate reader that simultaneously detects multiple analytes, it is possible to accelerate the amyloid fibril formation reaction from various amyloid-causing proteins and detect them with high throughput in a short period of time (Patent Document 1). Furthermore, by attaching an ultrasound irradiation device to a fluorescence spectrophotometer, it is possible to accelerate the amyloid fibril formation reaction and track the process in real time (Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2012 / 017739 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-246012 Summary of the Invention [Problem to be solved by the invention]

[0005] However, ultrasound irradiation devices are expensive, consume a lot of power, often emit noise, and have limitations on installation conditions, which makes the entire device large. An object of the present disclosure is to provide a novel method for forming amyloid fibrils that can form amyloid fibrils more easily than conventional methods. [Means for solving the problem]

[0006] As a result of extensive research aimed at solving the above problems, the present inventors have discovered that amyloid fibrils can be formed from amyloid fibril-forming proteins by moving a fluid containing the amyloid fibril-forming proteins in a specific manner.

[0007] That is, the gist of the present disclosure is as follows. [1] A method for forming amyloid fibrils from an amyloid fibril-forming protein, comprising: A fluid containing the amyloid fibril-forming protein is introduced into a fluid tube; A method for forming amyloid fibrils from the amyloid fibril-forming protein by transporting the fluid through a fluid tube by applying pressure and pressure to the fluid through the fluid tube and moving the fluid by gradually compressing the fluid tube in one direction. [2] The method according to [1], wherein one end of the fluid tube is connected to the other end, and the fluid is transported so as to circulate. [3] The method according to [1] or [2], wherein the fluid is conveyed at a rate of 0.0001 mL / min or more. [4] The method according to any one of [1] to [3], wherein the amyloid fibril-forming protein is α-synuclein, amyloid β, β2-microglobulin, or hen egg white lysozyme. [5] A fluid tube into which a fluid containing a protein that forms amyloid fibrils is introduced; means for applying pressure and pressure to the fluid through the fluid tube by progressively compressing the fluid tube in one direction to move the fluid; Amyloid fibril formation machinery. [6] The amyloid fibril formation apparatus according to [5], further comprising a means for detecting the amyloid fibrils. [7] The amyloid fibril formation device according to [6], wherein the amyloid fibrils detected by the means for detecting amyloid fibrils are amyloid fibrils formed from proteins that form the amyloid fibrils by transporting the fluid by the means for moving the fluid. [8] The amyloid fibril formation device according to [6] or [7], wherein the fluid contains an amyloid-specific fluorescent substance, and the means for detecting amyloid fibrils is capable of detecting fluorescence derived from the amyloid-specific fluorescent substance. [9] A fluid tube for transporting a fluid containing a protein that forms amyloid fibrils; means for applying pressure and pressure to the fluid through the fluid tube by progressively compressing the fluid tube in one direction to move the fluid; and means for detecting the amyloid fibrils. Amyloid fibril analysis system.

[10] The amyloid fiber analysis system according to [9], wherein the fluid contains an amyloid-specific fluorescent substance, and the means for detecting amyloid fibers is capable of detecting fluorescence derived from the amyloid-specific fluorescent substance. [Effects of the Invention]

[0008] According to the present disclosure, amyloid fibrils can be formed more easily than in the past. More specifically, compared to methods using ultrasound irradiation, power consumption is reduced and almost no noise is generated during operation, making it possible to use the device in medical institutions, particularly near patients, and the entire device can be made smaller, allowing amyloid fibrils to be formed inexpensively. Furthermore, compared to ultrasound, the stimulus caused by fluid movement is thought to be closer to the stimulus applied when amyloid fibrils are formed in vivo, making it possible to form amyloid fibrils under conditions closer to those in vivo. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of an amyloid fiber analysis apparatus according to an embodiment of the present disclosure. [Figure 2] 1 is a graph showing the fluorescence intensity at 485 nm when (a) 0.8 mL or (b) 3.0 mL of HEWL solution was delivered. [Figure 3] This is a fluorescence microscope image of HEWL amyloid fibrils (photograph substitute for drawing). [Figure 4] This is an electron microscope image of HEWL amyloid fibrils (photograph instead of drawing). [Figure 5] Graph showing the fluorescence intensity at 485 nm when (a) 0.3 mL, (b) 0.6 mL, (c) 2 mL, or (d) 8 mL of αSN solution was delivered. [Figure 6] This is a fluorescence microscope image of αSN amyloid fibrils (photograph instead of drawing). [Figure 7] This is an electron microscope image of αSN amyloid fibrils (photograph instead of drawing). [Figure 8] 10 is a graph showing the fluorescence intensity at 485 nm when an Aβ solution is delivered. [Figure 9] This is an electron microscope image of Aβ amyloid fibrils (photograph instead of drawing). [Figure 10] 1 is a graph showing the fluorescence intensity at 485 nm when a β2m solution (pH 1.8) was transported. [Figure 11]This is an electron microscope image of β2m amyloid fibrils formed from β2m solution (pH 1.8) (photograph instead of drawing). [Figure 12] 1 is a graph showing the fluorescence intensity at 485 nm when a β2m+SDS solution (pH 7.0) was transported. [Figure 13] This is an electron microscope image of β2m amyloid fibrils formed by transporting β2m + SDS solution (pH 7.0) (photograph substitute for drawing). [Figure 14] 1 is a graph showing the fluorescence intensity at 485 nm when a β2m-SDS solution (pH 7.0) was transported. [Figure 15] This is an electron microscope image of β2m amyloid fibrils formed by transporting β2m-SDS solution (pH 7.0) (photograph substitute for drawing). [Figure 16] This is an electron microscope image of β2m amyloid fibrils formed by ultrasonic treatment of a β2m + SDS solution (pH 7.0) (photograph substitute for drawing). DETAILED DESCRIPTION OF THE INVENTION

[0010] Each configuration and combination thereof in each embodiment is merely an example, and additions, omissions, substitutions, and other modifications of configurations are possible as appropriate within the scope of the present disclosure. The present disclosure is not limited by the embodiments, but only by the scope of the claims. Furthermore, each aspect disclosed in this specification can be combined with any other feature disclosed in this specification. Furthermore, in this disclosure, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits, and "A to B" means greater than or equal to A and less than or equal to B.

[0011] One embodiment of the present disclosure is a method for forming amyloid fibrils from an amyloid fibril-forming protein, comprising: A fluid containing the amyloid fibril-forming protein is introduced into a fluid tube; The method comprises gradually compressing the fluid tube in one direction, thereby conveying the fluid through the fluid tube by a means for applying pressure and pressure to the fluid to move the fluid, thereby forming the amyloid fibrils from the amyloid fibril-forming protein.

[0012] Amyloid fibrils are a state of matter similar to solute crystals. When a causative protein (amyloid fibril-forming protein) exists in a supersaturated state in a solvent, if a stimulus that overcomes the energy barrier is applied, the supersaturation state is resolved and seed amyloid is formed. Subsequently, the causative protein aggregates from the seed amyloid, forming amyloid fibrils. The formation of seed amyloid is thought to involve various factors, such as the interface with air, the interface with the container wall, and debris in the solution. However, the formation of amyloid fibrils by transporting a fluid containing an amyloid fibril-forming protein using the above-mentioned means has not been reported to date.

[0013] Examples of amyloid fibril-forming proteins that can be used include, but are not limited to, α-synuclein, which causes Parkinson's disease; amyloid β, which causes Alzheimer's disease; β2-microglobulin, which causes dialysis-related amyloidosis; IAPP (Islet amyloid polypeptide), which causes type II diabetes; antibody L chain, which causes AL amyloidosis; transthyretin, which causes senile systemic amyloidosis; apolipoprotein AI; apolipoprotein AII; gelsolin; lysozyme; fibrinogen α chain; serum amyloid A; immunoglobulin H chain; prion protein; cystatin; ABri precursor protein; ADan precursor protein; procalcitonin; amylin; ANP; prolactin; insulin; lactoferrin; keratoepithelin; lactadherin; and the like. Lysozymes include hen egg white lysozyme. The amyloid fibril-forming protein is preferably α-synuclein, amyloid β, β2-microglobulin, or hen egg white lysozyme. The amyloid β may be amyloid β1-40, amyloid β1-42, or amyloid β1-43, and is preferably amyloid β1-40.

[0014] The fluid containing the amyloid fibril-forming protein is usually an aqueous solution containing the amyloid fibril-forming protein, and is preferably a supersaturated solution of the amyloid fibril-forming protein.

[0015] The composition of the fluid is not particularly limited, and any known composition for forming amyloid fibrils from an amyloid fibril-forming protein can be selected. The concentration of the amyloid fibril-forming protein in the fluid is preferably 0.05 to 20.0 mg / mL, more preferably 0.10 to 1.00 mg / mL, from the viewpoint of facilitating the formation of amyloid fibrils.

[0016] The fluid may further contain a buffer to facilitate adjustment of the pH of the fluid. The buffer is not particularly limited, but for example, a sodium phosphate buffer or a sodium citrate buffer can be used to facilitate adjustment of the pH of the fluid within a preferred range. The concentration of the buffer in the fluid is not particularly limited, but can be, for example, 5 mM to 500 mM.

[0017] The pH of the fluid can be appropriately selected depending on the type of amyloid fibril-forming protein so that the amyloid fibril formation reaction can proceed easily. It is preferably 1.8 to 9.0, and may be 5.0 to 8.0, or 5.5 to 7.0. When the pH of the fluid is within the above range, the amyloid fibril formation reaction can proceed easily. In this specification, the pH is measured at 25°C.

[0018] The fluid may comprise an amyloid-specific fluorescent substance, such as thioflavin T and thioflavin S, with thioflavin T being preferred.

[0019] The fluid may contain a surfactant depending on the type of amyloid fibril-forming protein. Examples of surfactants include sodium dodecyl sulfate (SDS). The inclusion of a surfactant in the fluid may promote the formation of amyloid fibrils. Specifically, Yamamoto et al., Biochemistry, 43, 11075-11082, reported that the inclusion of approximately 0.5 mM SDS in a solution promotes the formation of amyloid fibrils from β2-microglobulin. On the other hand, according to the method of this embodiment, amyloid fibrils can be formed even without a surfactant. Since it is difficult to imagine an environment in a living body where surfactants at the above concentrations exist, the method of this embodiment allows amyloid fibrils to be formed under conditions closer to those in a living body.

[0020] The fluid tube used in this embodiment is generally cylindrical, and allows the fluid to flow inside. It is preferable that at least a portion of the fluid tube has flexibility so that it can deform in response to pressure applied from the outside. Examples of materials for the flexible fluid tube include elastomers. Specific examples include fluororesin, polyolefin, polyurethane, silicone, and polyvinyl chloride.

[0021] Furthermore, it is preferable that at least a portion of the fluid tube is permeable to the fluorescence derived from the amyloid-specific fluorescent substance. Examples of materials for the fluid tube that are permeable include glass and polyvinyl chloride. Furthermore, at least a portion of the fluid tube may be constituted by a flow cell.

[0022] The fluid tube may be a single tube or a combination of two or more tubes, which may be made of the same material or different materials.

[0023] The inner diameter of the fluid tube is not particularly limited, but is preferably 0.5 to 3.0 mm, more preferably 0.7 to 2.0 mm, and particularly preferably 0.8 to 1.5 mm. When the inner diameter of the fluid tube is within the above range, amyloid fibers are easily formed. When the fluid tube is composed of two or more tubes, any one of the two or more tubes may be within the above range, or all of the two or more tubes may be within the above range.

[0024] The length of the fluid tube is not particularly limited, but is preferably 0.1 to 10 m, more preferably 0.2 to 5 m, and particularly preferably 0.5 to 3 m. When the fluid tube is made up of two or more tubes, the total length of the two or more tubes may be within the above range.

[0025] In this embodiment, a means is used for gradually compressing the fluid tube in one direction to apply pressure and pressure to the fluid through the fluid tube, thereby moving the fluid. In a preferred embodiment, the means may repeatedly apply gradual compression in one direction to a portion of the fluid tube. In this way, the movement of the fluid becomes a pulsating flow.

[0026] "Progressive compression in one direction" may mean, for example, compressing a portion of the fluid tube in the circumferential direction and then continuously moving the compression position in the axial direction of the fluid tube. This causes the fluid to be pressurized on the side of the compression position in the direction of movement and decompressed on the side opposite to the direction of movement of the compression position. The number of compression positions may be one or more, and is preferably two or more.

[0027] The means for gradually compressing the fluid tube in one direction to apply pressure and pressure to the fluid through the fluid tube and move the fluid may be, for example, a peristaltic pump (also called a roller pump or a tube pump). The power consumption of a peristaltic pump is approximately 2.8 to 6 W, which is approximately 10 times lower than that of an ultrasound irradiation device. Furthermore, when a peristaltic pump is used, the movement of the fluid becomes a pulsating flow. Furthermore, while ultrasound cannot avoid noise during operation, this method operates quietly, making it possible to use it in clinical institutions and near patients.

[0028] In this embodiment, one end of the fluid tube may be connected to the other end. This allows the fluid to be transported in a circulating manner. When the fluid is circulated, the fluid is pressurized or depressurized by the means for moving the fluid each time the fluid makes a circuit of the fluid tube. This stimulation can promote the formation of amyloid fibrils. Furthermore, the formation of amyloid fibrils can be continuously monitored by the means for detecting amyloid fibrils, which will be described later.

[0029] The fluid transport speed is not particularly limited, but is preferably 0.0001 mL / min or more, more preferably 0.001 mL / min or more, even more preferably 0.01 mL / min or more, and particularly preferably 0.1 mL / min or more. It is also preferably 1000 mL / min or less, more preferably 100 mL / min or less, and particularly preferably 10 mL / min or less. That is, the fluid transport speed may be 0.0001 to 1000 mL / min, 0.001 to 100 mL / min, 0.01 to 10 mL / min, or 0.1 to 10 mL / min. The fluid transport speed is an average speed and can be calculated by measuring the volume of fluid flowing out per minute.

[0030] In this embodiment, the formed amyloid fibrils may be detected by a means for detecting amyloid fibrils. The means for detecting amyloid fibrils is not particularly limited and may be a means for directly detecting amyloid fibrils or a means for indirectly detecting amyloid fibrils. Examples of means for directly detecting amyloid fibrils include electron microscopes, light scattering detectors, and absorptiometers. Examples of electron microscopes include transmission electron microscopes and scanning electron microscopes. Furthermore, the characteristics of amyloid fibrils can be evaluated in detail by detecting light scattering, changes in absorbance, and the like using light scattering detectors and absorptiometers. Examples of means for indirectly detecting amyloid fibrils include those capable of detecting fluorescence derived from amyloid-specific fluorescent substances, and specifically include fluorescence microscopes and fluorometers.

[0031] Another embodiment of the present disclosure is a fluid tube into which a fluid containing an amyloid fibril-forming protein is introduced; means for applying pressure and pressure to the fluid through the fluid tube by progressively compressing the fluid tube in one direction to move the fluid; It is an amyloid fibril formation device.

[0032] The above explanations are applicable to the fluid containing the amyloid fibril-forming protein, the fluid tube, and the means for moving the fluid. The amyloid fibril formation device according to this embodiment may also include a means for detecting the amyloid fibrils.

[0033] Furthermore, yet another embodiment of the present disclosure is a fluid tube for transporting a fluid containing a protein that forms amyloid fibrils; means for applying pressure and pressure to the fluid through the fluid tube by progressively compressing the fluid tube in one direction to move the fluid; and means for detecting the amyloid fibrils. This is an amyloid fibril analysis system.

[0034] An amyloid fiber analysis device 100 (hereinafter, sometimes referred to as "device 100") will be described below as an example of an amyloid fiber analysis system according to this embodiment, with reference to Fig. 1. Note that the following configuration is an example, and the present disclosure is not limited thereto.

[0035] The device 100 includes a fluid tube 1 that transports a fluid containing an amyloid fibril-forming protein. For example, the fluid tube 1 forms a loop, allowing the fluid to circulate. For example, the fluid containing the amyloid fibril-forming protein is introduced into the fluid tube 1 via an injection valve 2. For example, the injection valve 2 has an inlet that can be opened or closed. After the fluid flows into the fluid tube through the inlet, the inlet can be closed, thereby forming the fluid tube 1 into a closed loop. For another example, the injection valve 2 has an inlet and an injection loop (not shown) that can be opened or closed. Here, the injection valve 2 is configured to be switchable between a state in which one end of the injection loop is connected to one end of the fluid tube 1 and the other end of the injection loop is connected to the other end of the fluid tube 1, and a state in which one end of the injection loop is connected to the other end of the injection loop and one end of the fluid tube 1 is connected to the other end of the fluid tube 1. In this case, the fluid can be injected from the injection port into the injection loop, and then the flow path can be switched so that the fluid flows from the injection loop into the fluid tube 1. Alternatively, the fluid containing the amyloid fibril-forming protein may be introduced directly from one end of the fluid tube 1 without passing through the injection valve 2. In this case, for example, with one end of the fluid tube 1 immersed in the fluid, The fluid can be made to flow into the fluid tube 1 by driving the peristaltic pump 3, which will be described later.

[0036] The fluid that has flowed into the fluid tube 1 is pumped by a peristaltic pump 3. The peristaltic pump 3 has a mounting portion to which a portion of the fluid tube 1 can be attached, and one or more rollers that limit the volume of the mounting portion. By adjusting the position of the rollers relative to the fluid tube 1 attached to the mounting portion, the fluid tube 1 can be compressed in the circumferential direction. In this state, by moving the rollers in the axial direction of the fluid tube 1, the fluid tube 1 is gradually compressed in the axial direction. As a result, the fluid that has flowed into the fluid tube 1 is pressurized and depressurized through the fluid tube, and moves in the axial direction of the fluid tube 1.

[0037] Although the device 100 includes a fluorometer 4 and a fluorescence microscope 6, the device is not limited thereto, and an amyloid fiber analysis system according to an embodiment of the present disclosure may not include one or both of the fluorometer 4 and the fluorescence microscope 6. The fluid tube 1 may be configured to pass through the measurement unit of the fluorometer 4 and the observation unit of the fluorescence microscope 6. It is preferable that at least the portion of the fluid tube 1 that passes through the measurement unit of the fluorometer 4 and the observation unit of the fluorescence microscope 6 is transparent. When the fluid contains an amyloid-specific fluorescent substance such as thioflavin T, the fluorescence from the fluorescent substance can be measured using a fluorometer 4. Furthermore, the morphology of the amyloid to which the fluorescent substance is attached can be observed using a fluorescence microscope 6.

[0038] The apparatus 100 may further include a light scattering detector, a refractive index detector, and / or an absorptiometer, which allows for detailed characterization of the amyloid fibrils.

[0039] An amyloid fiber analysis system according to an embodiment of the present disclosure may recover and detect amyloid fibers from the fluid tube 1. In this case, a means for detecting amyloid fibers, such as the fluorometer 4 and / or the fluorescence microscope 6, does not need to be attached to the fluid tube 1. Furthermore, instead of the fluorometer 4 and the fluorescence microscope 6, an electron microscope or the like may be used to detect amyloid fibers.

[0040] The device 100 includes an air trap 5, but is not limited to this and may not include an air trap 5. If the air trap 5 is included, air bubbles generated in the fluid tube 1 can be removed.

[0041] 1, an injection valve 2, a peristaltic pump 3, a fluorometer 4, an air trap 5, and a fluorescence microscope 6 are arranged in this order along the axial direction of a fluid tube 1, but this order is not limited to this, and the arrangement of each component may be interchanged, or each component may be arranged multiple times. In a preferred embodiment, the fluorometer 4 and / or the fluorescence microscope 6 are arranged downstream of the peristaltic pump 3 (in the direction of fluid transport). [Example]

[0042] The present disclosure will be specifically described below with reference to examples, but the present disclosure is not limited to the aspects of the following examples.

[0043] <Amyloid fibril-forming proteins and compounds used> Recombinant human α-synuclein (hereinafter sometimes referred to as "αSN") was expressed in Escherichia coli and purified by the method described in Yagi et al., J. Biol. Chem., 280, 38609-38616 (2005). Recombinant human β2 microglobulin (hereinafter referred to as "β2m") with a methionine residue added to the N-terminus was expressed in Escherichia coli and purified using the method described in Chiba et al., J. Biol. Chem., 278, 47016-47024 (2016). Hen egg white lysozyme (hereinafter sometimes referred to as "HEWL") was purchased from Nacalai Tesque. Amyloid β1-40 (hereinafter sometimes referred to as "Aβ") was purchased from Peptide Institute, Inc. The fluorescent dye thioflavin T (hereinafter sometimes referred to as "ThT") was purchased from Wako Pure Chemical Industries. All other reagents were purchased from Nacalai Tesque.

[0044] Experiment 1. Peristaltic pump-dependent formation of HEWL amyloid fibrils HEWL was dissolved in deionized water and added to a buffer solution containing 20 mM HCl, 2 M guanidinium chloride, and 5 μM ThT to prepare a 1.0 mg / mL HEWL solution (pH 1.8). An amyloid fibril formation assay was performed using the apparatus shown in Figure 1. Monotarou silicone tubing (MGJG10-1.5, inner diameter 1.0 mm) was used as the fluid tubing. An ISMATEC pump (REGLO Analog MS-2 / 12) was used as the peristaltic pump. A JASCO FP920 spectrofluorometer was used as the fluorometer. A 16 μL flow cell was used for the fluorometer. 0.8 mL or 3.0 mL of 1.0 mg / mL HEWL solution was introduced into the fluid tube at a flow rate of 0.1 mL / min, and then both ends of the fluid tube were connected to form a loop. The total length of the loop was approximately 1 m, and the total volume of the fluid was 2–3 mL. The peristaltic pump was then operated at a flow rate of 0.1 mL / min. The fluorescence intensity at 485 nm measured with a fluorometer is shown in FIG.

[0045] As shown in Figure 2, when 0.8 mL and 3.0 mL of HEWL solution were injected, peaks of fluorescence intensity appeared approximately every 20 minutes, and the baseline intensity gradually increased. Initially, ThT was localized in a portion of the fluid tubing, and peaks appeared as these passed through the flow cell. However, as time passed, ThT dispersed, and the peaks disappeared. Meanwhile, amyloid fibrils formed over time, and ThT attached to them, resulting in a gradual increase in the baseline intensity.

[0046] Amyloid fibrils were formed in the same manner as above, except that 1.5 mL of a 2.0 mg / mL HEWL solution was infused at a flow rate of 0.1 mL / min. A 1.3 mm diameter glass tube was used as the fluid tube for observation under a fluorescence microscope. Figure 3 shows an image (110 minutes after the start of the assay) obtained using a Keyence fluorescence microscope with a BX filter and GFP. As shown in Figure 3, ThT-positive aggregates were observed.

[0047] To confirm that the ThT-positive aggregates were amyloid fibrils, the solution was collected after the assay and observed under a transmission electron microscope (TEM, JEM-1400, JEOL Ltd.). Specifically, 10 μL of the solution after observation was placed on a collodion-coated mesh (Cu, 300 mesh, Nissin EM Co., Ltd.) for 1 minute. After removing excess liquid, the grid was stained with a 1% (w / v) uraniur acetate solution for 1 minute. The surface of the grid was then rinsed with deionized water. The grid was then observed at an accelerating voltage of 80 kV. The observed image is shown in Figure 4.

[0048] As shown in Figure 4, rigid amyloid fibrils with a diameter of 10 nm and a length of 1 μm were observed.

[0049] Next, instead of the ISMATEC pump, an ATTO Peristaltic Bio Mini Pump (AC-2120) and an AS ONE Tubing Pump (TP-10SA) were used as peristaltic pumps. The flow rate was set to the minimum speed for each speed, and the discharged flow rate was measured. The results are shown in Table 1. The flow rate was 1.5 mL of a 2.0 mg / mL HEWL solution as the fluid containing the amyloid fibril-forming protein, and amyloid fibrils were formed in the same manner as above. The tubes attached to the pump had the inner diameters shown in Table 1, but tubes with an inner diameter of 1.0 mm and a length of 2.0 m were used for other parts. ThT-positive aggregates were observed under a fluorescence microscope when either pump was used, suggesting that amyloid fibrils had formed. When the fluorescence was observed using a fluorescence microscope, different pulsating flow patterns were observed depending on the pump, as shown in Table 1. The above results demonstrate that amyloid fibrils are formed by transporting a fluid using a method according to an embodiment of the present disclosure, regardless of the pulsating flow pattern. [Table 1]

[0050] Experiment 2. Formation of αSN amyloid fibrils αSN was dissolved in deionized water and added to a buffer solution containing 10 mM sodium phosphate, 0.5 M sodium sulfate, and 5 μM ThT to prepare a 0.5 mg / mL αSN solution (pH 7.0). An amyloid fibril formation assay was performed using the apparatus shown in Figure 1. An ISMATEC pump (REGLO Analog MS-2 / 12) was used as the peristaltic pump. A JASCO FP920 spectrofluorometer was used as the fluorometer. A Keyence fluorescence microscope was used as the fluorescence microscope, using a BX filter GFP. 0.3 mL, 0.6 mL, 2 mL, or 8 mL of 0.5 mg / mL αSN solution was flowed into the fluid tube at a flow rate of 0.1 mL / min. Other conditions were the same as in Experiment 1.

[0051] The fluorescence intensity at 485 nm measured with a fluorometer is shown in Figure 5. Images observed with a fluorescence microscope are shown in Figure 6. Furthermore, the solution after the assay was observed with a TEM in the same manner as above, and the results are shown in Figure 7. As shown in Figure 5, amyloid fibrils formed over time, and ThT attached to these, which is thought to have gradually increased the baseline intensity. Furthermore, fluffy aggregates were observed, as shown in Figures 6 and 7. This demonstrates that amyloid fibrils of αSN are formed by the method according to an embodiment of the present disclosure.

[0052] Experiment 3. Amyloid fibril formation of Aβ Aβ was dissolved in 0.05% (w / w) aqueous ammonia solution and added to a buffer solution containing 10 mM sodium phosphate, 0.1 M NaCl, and 5 μM ThT to obtain a 0.1 mg / mL Aβ solution (pH 7.0). An amyloid fibril formation assay was carried out in the same manner as in Experiment 2. 1.0 mL of Aβ solution was flowed into the fluid tube at a flow rate of 0.1 mL / min. The fluorescence intensity at 485 nm measured with a fluorometer is shown in Figure 8. Furthermore, the solution after the assay was observed by TEM in the same manner as above, and the results are shown in Figure 9. Figures 8 and 9 demonstrate that Aβ amyloid fibrils are formed by the method according to an embodiment of the present disclosure.

[0053] In addition, an amyloid fibril formation assay was performed in the same manner as above, except that Aβ was dissolved in DMSO and then added to the Aβ buffer. In this case, Aβ amyloid fibrils were formed, as in the above.

[0054] Experiment 4. Formation of β2m amyloid fibrils β2m was dissolved in deionized water and further diluted with 20 mM HCl, 0.4 M NaCl, 5 μM It was added to a buffer solution containing ThT to obtain a 0.5 mg / mL β2m solution (pH 1.8). An amyloid fibril formation assay was carried out in the same manner as in Experiment 2. 1.0 mL of β2m solution was flowed into the fluid tube at a flow rate of 0.1 mL / min. The fluorescence intensity at 485 nm measured with a fluorometer is shown in Figure 10. Furthermore, the solution after the assay was observed by TEM in the same manner as above, and the results are shown in Figure 11. Figures 10 and 11 demonstrate that β2m amyloid fibrils are formed under acidic conditions by the method according to an embodiment of the present disclosure.

[0055] Next, β2m was dissolved in deionized water and added to a buffer containing 10 mM sodium phosphate, 0.1 M NaCl, 0.5 mM SDS, and 5 μM ThT to obtain a 20 mg / mL β2m solution (pH 7.0). Hereinafter, this solution will be referred to as the β2m + SDS solution. An amyloid fibril formation assay was carried out in the same manner as in Experiment 2. 1.5 mL of β2m solution was flowed into the fluid tube at a flow rate of 0.1 mL / min. The fluorescence intensity at 485 nm measured with a fluorometer is shown in Figure 12. Furthermore, the solution after the assay was observed by TEM in the same manner as above, and the results are shown in Figure 13. Figures 12 and 13 demonstrate that amyloid fibrils of β2m are formed under neutral conditions in the presence of SDS by a method according to an embodiment of the present disclosure.

[0056] A 20 mg / mL β2m solution (pH 7.0) was obtained in the same manner as above, except that 0.5 mM SDS was not added. Hereinafter, this solution will be referred to as the β2m-SDS solution. An amyloid fibril formation assay was carried out in the same manner as in Experiment 2. 1.5 mL of β2m solution was flowed into the fluid tube at a flow rate of 0.1 mL / min. The fluorescence intensity at 485 nm measured with a fluorometer is shown in Figure 14. Furthermore, the solution after the assay was observed by TEM in the same manner as above, and the results are shown in Figure 15. Figures 14 and 15 demonstrate that amyloid fibrils of β2m are formed under neutral conditions in the absence of SDS by the method according to an embodiment of the present disclosure.

[0057] The above β2m+SDS solution or β2m-SDS solution was subjected to ultrasonic irradiation using a Hitachi F-7000 spectrofluorometer. 2 mL of the above solution was placed in a cuvette with a 1 cm optical path length. While stirring the solution with a stirrer rotating at 600 rpm, ultrasonic treatment was performed for 1 minute at 2-minute intervals. The frequency was 27.5 kHz, and the output was 0.14 W. The temperature was 25°C.

[0058] When the β2m+SDS solution was sonicated, the fluorescence intensity increased, suggesting the formation of amyloid fibrils. After approximately 3 hours of treatment, the solution was observed by TEM in the same manner as above, and the results are shown in Figure 16. It was confirmed that sonication of the β2m+SDS solution resulted in the formation of amyloid fibrils.

[0059] On the other hand, when a β2m-SDS solution was subjected to ultrasonic treatment, the formation of amyloid fibrils was not confirmed. That is, according to the method according to an embodiment of the present disclosure, amyloid fibrils can be formed even under conditions where ultrasonic treatment does not form amyloid fibrils. Since it is difficult to imagine an environment in which a surfactant such as 0.5 mM SDS is present, a method according to an embodiment of the present disclosure may be able to form amyloid fibrils under conditions closer to those in vivo than ultrasonic treatment. [Explanation of symbols]

[0060] 100 Amyloid Fibril Analyzer 1 fluid tube 2 injection valve 3 Peristaltic Pump 4. Fluorometer 5 Air Trap 6. Fluorescence Microscopy

Claims

1. 1. A method for forming amyloid fibrils from an amyloid fibril-forming protein, comprising: A fluid containing the amyloid fibril-forming protein is introduced into a fluid tube; A method for forming amyloid fibrils from the amyloid fibril-forming protein by transporting the fluid through a fluid tube by applying pressure and pressure to the fluid through the fluid tube and moving the fluid by gradually compressing the fluid tube in one direction.

2. The method according to claim 1 , wherein one end of the fluid tube is connected to the other end, and the fluid is conveyed in a circulating manner.

3. The method according to claim 1 or 2, wherein the fluid is delivered at a rate of 0.0001 mL / min or more.

4. The method according to claim 1 or 2, wherein the amyloid fibril-forming protein is α-synuclein, amyloid β, β2 microglobulin, or hen egg white lysozyme.

5. a fluid tube into which a fluid containing a protein that forms amyloid fibrils is introduced; means for applying pressure and pressure to the fluid through the fluid tube by progressively compressing the fluid tube in one direction to move the fluid; Amyloid fibril formation machinery.

6. The amyloid fibril formation apparatus according to claim 5 , further comprising a means for detecting the amyloid fibrils.

7. 7. The amyloid fibril formation device according to claim 6, wherein the amyloid fibrils detected by the means for detecting amyloid fibrils are amyloid fibrils formed from proteins that form the amyloid fibrils by transporting the fluid by the means for moving the fluid.

8. 8. The amyloid fibril formation device according to claim 6, wherein the fluid contains an amyloid-specific fluorescent substance, and the means for detecting amyloid fibrils is capable of detecting fluorescence derived from the amyloid-specific fluorescent substance.

9. a fluid tube for transporting a fluid containing a protein that forms amyloid fibrils; means for applying pressure and pressure to the fluid through the fluid tube by progressively compressing the fluid tube in one direction to move the fluid; and means for detecting the amyloid fibrils. Amyloid fibril analysis system.

10. 10. The amyloid fiber analysis system according to claim 9, wherein the fluid contains an amyloid-specific fluorescent substance, and the means for detecting the amyloid fibers is capable of detecting fluorescence derived from the amyloid-specific fluorescent substance.

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