Method for detecting misfolded proteins in biological samples

Abstract

To provide a method for rapidly and easily detecting amyloid in biological samples.SOLUTION: Provided is a method for detecting misfolded proteins in biological samples, that includes the steps of: irradiating a biological sample with excitation light of a specific excitation wavelength; and detecting self-fluorescence emitted from the misfolded proteins according to irradiation of the excitation light.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting misfolded proteins, a method for assisting in the diagnosis of amyloidosis, and an apparatus for detecting misfolded proteins. [Background technology]

[0002] Amyloids are abnormal fibrous proteins formed by protein misfolding. Due to their cross-β sheet structure, amyloids have properties that differ from normal biological proteins, such as specific binding to Congo red dye and high resistance to heat and enzyme treatment.

[0003] When amyloid accumulates locally or throughout the body, it causes amyloidosis, which damages various organs, including the brain. Amyloidosis is a progressive, intractable disease in humans and animals, and 36 types of amyloidosis, including Alzheimer's disease and Parkinson's disease, are known, each for a different causative protein. In recent years, the number of amyloidosis patients has been increasing year by year, and there is a demand for technology that enables early diagnosis.

[0004] Detection of amyloid is also an important issue in meat hygiene. Recent studies have revealed that amyloid can be transmitted between different species, and it is known that the consumption of meat containing amyloid can be a risk factor for the development of amyloidosis (Non-Patent Document 1). Therefore, there is a demand for an efficient method for testing amyloid in meat.

[0005] At present, the histopathological examination method (pathological examination), which has been used for more than 100 years, is mainly used for detecting amyloid (Non-Patent Document 2). In this method, tissues extracted from a living body by biopsy or autopsy are fixed in formalin, embedded in paraffin, sliced ​​and stained into tissue sections, and then an experienced pathologist observes the sample under an optical microscope. Normal pathological diagnosis is performed by hematoxylin-eosin staining, but identification of amyloid requires proof by Congo red staining in addition to this staining. The use of the histopathological examination method is greatly limited by the fact that it requires proficiency in histopathology. Furthermore, since the test itself takes about 3 days to 1 week, it is also problematic that amyloid cannot be detected quickly. In recent years, various dyes for staining amyloid other than Congo red have been developed, but they are the same in that they label and detect amyloid, and the problems of requiring proficiency in histopathology and low throughput have not yet been overcome.

[0006] Positron emission tomography (Amyloid PET) is another method other than histopathological examination. This method uses a radioisotope probe that specifically binds to amyloid to image the distribution of amyloid deposits in the body. The advantages of Amyloid PET are that the test results can be obtained within a few hours and that invasiveness is kept to a minimum, but there are many disadvantages in its implementation, such as the need for sufficient skill in the evaluation, the need for large-scale equipment, and the high cost of the test.

[0007] Therefore, there is a need for a method for quickly and easily detecting amyloid in a biological sample that overcomes the above-mentioned problems of the prior art. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Murakami T, Ishiguro N, Higuchi K. Transmission of Systemic AA Amyloidosis in Animals. Veterinary Pathology 51(2): 363-371, 2014. [Non-Patent Document 2] Kadota A, Iwaide S, Miyazaki S, Mitsui I, Machida N, Murakami T. Pathology and proteomics-based diagnosis of localized light-chain amyloidosis in dogs and cats. Veterinary Pathology 57(5): 658-665, 2020. Summary of the Invention [Problem to be solved by the invention]

[0009] It is an object of the present invention to provide a method for rapid and easy detection of amyloid in a biological sample. [Means for solving the problem]

[0010] Fluorescence fingerprint analysis is a technique that brute-force detects the fluorescence intensity of a substance in any excitation / fluorescence wavelength range, and obtains three-dimensional information consisting of the excitation wavelength, fluorescence wavelength, and fluorescence intensity. Fluorescence fingerprint analysis is used in the food industry as an ingredient analysis technique that identifies specific ingredients without labeling. However, there are no known examples of fluorescence fingerprint analysis being applied to pathological analysis.

[0011] In order to solve the above problems, the present inventors prepared homogenates from bovine livers containing amyloid and performed fluorescence fingerprint analysis under non-labeled conditions, contrary to conventional technical knowledge. As a result, they found two prominent autofluorescence peaks contained only in amyloid-positive livers, and demonstrated that amyloid can be detected quickly and easily without labeling based on the autofluorescence peaks. The present invention is based on the above new findings and provides the following.

[0012] (1) A method for detecting a misfolded protein in a biological sample, the method comprising the steps of irradiating the biological sample with excitation light having a specific excitation wavelength, and detecting autofluorescence emitted from the misfolded protein upon irradiation with the excitation light. (2) The method according to (1), wherein the misfolded protein is an amyloid. (3) The method according to (2), wherein the specific excitation wavelength is 320 nm to 380 nm and / or 420 nm to 480 nm. (4) The method according to (3), wherein the fluorescence wavelength of the autofluorescence detected in the detection step is 400 nm to 510 nm. (5) A method for detecting a misfolded protein in a biological sample, comprising the steps of: irradiating the biological sample with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range; measuring the wavelength and intensity of autofluorescence emitted from the biological sample upon irradiation with the excitation light; and determining the presence or absence of a misfolded protein that emits autofluorescence based on the obtained measured values. (6) The method according to (5), wherein the specific excitation wavelength range includes 320 nm to 380 nm and / or 420 nm to 480 nm. (7) The method according to (5) or (6), wherein the misfolded protein is an amyloid. (8) The method according to (7), wherein in the determination step, the biological sample is determined to contain amyloid if the following (a) and / or (b) is met: (a) the peak wavelength of the autofluorescence emitted from the biological sample when irradiated with excitation light having an excitation wavelength range including 320 nm to 380 nm is in the range of 400 nm to 460 nm, and / or (b) When the biological sample is irradiated with excitation light having a wavelength range of 420 nm to 480 nm, the peak wavelength of the autofluorescence emitted from the biological sample is in the range of 450 nm to 510 nm. (9) The method according to any one of (1) to (8), wherein the biological sample is a tissue or a body fluid.

[0013] (10) A method for assisting in the diagnosis of amyloidosis, comprising the steps of: irradiating biological samples derived from a subject and a control with excitation light having a specific excitation wavelength; measuring the fluorescence intensity of autofluorescence emitted from amyloid in the biological samples by the irradiation with the excitation light; comparing the obtained fluorescence intensities between the subject and the control; and determining that the subject is affected by amyloidosis when the fluorescence intensity of the subject is higher than that of the control, wherein the specific excitation wavelength is 320 nm to 380 nm and / or 420 nm to 480 nm, and the fluorescence wavelength of the autofluorescence measured in the measuring step is 400 nm to 510 nm. (11) A method for assisting in the diagnosis of amyloidosis, comprising the steps of: irradiating a biological sample derived from a subject with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range; measuring the wavelength and intensity of autofluorescence emitted from the biological sample by the irradiation with the excitation light; and determining that the subject is affected by amyloidosis in the following cases (a) and / or (b) based on the obtained measurement values, wherein the specific excitation wavelength range includes 320 nm to 380 nm and / or 420 nm to 480 nm. (a) the peak wavelength of the autofluorescence emitted from the biological sample when irradiated with excitation light having an excitation wavelength range including 320 nm to 380 nm is in the range of 400 nm to 460 nm, and / or (b) When the biological sample is irradiated with excitation light having a wavelength range of 420 nm to 480 nm, the peak wavelength of the autofluorescence emitted from the biological sample is in the range of 450 nm to 510 nm. (12) The method according to (10) or (11), wherein the subject is a livestock animal. (13) The method according to any one of (10) to (12), wherein the amyloidosis is AA amyloidosis.

[0014] (14) An apparatus for detecting misfolded proteins in a biological sample, comprising: (a) a light source that emits excitation light having a wavelength of 320 nm to 480 nm; (b) an optical unit that separates fluorescence having a wavelength of 400 nm to 510 nm; and (c) a detection unit that detects autofluorescence emitted from the biological sample. (15) An apparatus for detecting misfolded proteins in a biological sample, comprising: (a) a light source that emits excitation light in an excitation wavelength range including 320 nm to 480 nm; (b) an optical unit that separates fluorescence in a fluorescence wavelength range including 400 nm to 510 nm; (c) a detection unit that detects autofluorescence emitted from the biological sample; and (d) a wavelength varying unit that changes the wavelength of the excitation light and / or fluorescence in a stepwise or continuous manner. Effect of the Invention

[0015] According to the present invention, a method for quickly and easily detecting amyloid in a biological sample is provided. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows a conventional histopathological amyloid detection method and the steps constituting one embodiment of the present invention. [Diagram 2] 2A and 2B show the results of a fluorescent fingerprint analysis of liver homogenates from amyloid-negative bovine individuals, in which FIG. 2A shows a contour plot of the fluorescent fingerprint analysis results and FIG. 2B shows a three-dimensional plot of the fluorescent fingerprint analysis results. [Diagram 3] 3 shows the results of fluorescence fingerprint analysis of liver homogenates from amyloid-positive bovine individuals, in which FIG 3A shows a contour plot of the fluorescence fingerprint analysis results and FIG 3B shows a three-dimensional plot of the fluorescence fingerprint analysis results. [Figure 4] 4 shows the results of a fluorescence fingerprint analysis of an amyloid form of LPS-binding protein, in which: Figure 4A shows a contour plot of the fluorescence fingerprint analysis result; Figure 4B shows a three-dimensional plot of the fluorescence fingerprint analysis result. [Diagram 5]5 shows the results of fluorescence fingerprint analysis of the amyloid form of Aβ42 peptide, in which: Figure 5A shows a contour plot of the fluorescence fingerprint analysis result; and Figure 5B shows a three-dimensional plot of the fluorescence fingerprint analysis result. [Figure 6] 6 shows the results of a fluorescence fingerprint analysis of the amyloid form of α-casein protein, in which: Figure 6A shows a contour plot of the fluorescence fingerprint analysis results; Figure 6B shows a three-dimensional plot of the fluorescence fingerprint analysis results. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 1. Misfolded protein detection method 1-1. Overview A first aspect of the present invention is a method for detecting a misfolded protein. The misfolded protein detection method of this embodiment includes an irradiation step and a detection step, or includes an irradiation step, a measurement step, and a determination step. According to the misfolded protein detection method of this embodiment, misfolded proteins in a biological sample can be detected quickly and easily without labeling.

[0018] 1-2.Definition of Terms The following terms frequently used in this specification are defined below. As used herein, the term "misfolded protein" refers to a protein that is not folded correctly compared to the original protein. A protein is a chain molecule in which multiple amino acids are linked in a one-dimensional manner, and a folded structure of the polypeptide chain including α-helix and β-sheet is formed by hydrogen bonds, hydrophobic bonds, ionic bonds, etc. between amino acid residues and water molecules, and the protein is stabilized in a certain three-dimensional structure. In a misfolded protein, at least a part of the folded structure of the polypeptide chain is lost, and the three-dimensional structure of the polypeptide is changed from the original three-dimensional structure. In a misfolded protein, the function or activity (e.g., catalytic activity) is often reduced or lost. In addition, misfolded proteins often aggregate due to abnormal folding, which can cause impairment of biological functions. Examples of misfolded proteins include amyloids, which will be described later, as well as CFTR (cystic fibrosis transmembrane conductance regulator) protein, which can be denatured in cystic fibrosis, and TDP-43 protein, which can be denatured in amyotrophic lateral sclerosis. Diseases caused by or accompanied by the denaturation or aggregation of polypeptides are called "protein-denaturing diseases." Examples of "protein-denaturing diseases" include amyloidosis, tauopathy, cystic fibrosis, and amyotrophic lateral sclerosis, which will be described later.

[0019] "Amyloid" is a fibrous protein characterized by a cross-β sheet structure that is generated by protein misfolding. The cross-β sheet structure (also called cross-β structure) is a structure in which β-sheet structures are regularly aligned perpendicular to the fiber axis, and is known to be commonly contained in amyloid fibrils regardless of the primary structure. Amyloid can be generated by misfolding of the full-length amyloid precursor protein or a partially cleaved or degraded amyloid precursor protein. Examples of amyloids include, but are not limited to, amyloid beta (sometimes referred to as "Aβ") derived from the amyloid beta precursor protein, amyloid A (sometimes referred to as "AA") derived from the serum amyloid A protein, amyloid derived from the alpha-synuclein protein (sometimes referred to as "AαSyn"), amyloid derived from the prion protein (PrP) (sometimes referred to as "APrP"), and amyloid derived from the huntingtin (Htt) protein, as well as amyloid derived from the Ig light chain (kappa, lambda) protein (sometimes referred to as "AL"), amyloid derived from the ApoAII protein (sometimes referred to as "AApoAII"), transthyrelin (trans-thyrelin), and the like. Known types of amyloid include amyloid derived from the tau protein (sometimes called "ATau"), amyloid derived from the amylin protein (sometimes called "AIAPP"), amyloid derived from insulin (sometimes called "AIns"), amyloid derived from alpha-casein protein (sometimes called "ACas"), amyloid derived from calcitonin protein (sometimes called "ACal"), and amyloid derived from odontogenic ameloblast-associated protein (sometimes called "AOAPP").

[0020] As used herein, the term "amyloid precursor protein" refers to a protein that can form the above-mentioned amyloid. Examples of amyloid precursor proteins include amyloid β precursor protein, serum amyloid A protein (e.g., SAA1 protein, SAA2 protein, and SAA4 protein described below), α-synuclein protein, prion protein (PrP), huntingtin (Htt) protein, Ig light chain (κ, λ) protein, ApoAII protein, transthyretin protein, fibrinogen protein, ApoAI protein, tau protein, amylin protein, insulin, α-casein protein, calcitonin protein, odontogenic ameloblast-associated protein, etc.

[0021] Amyloid β peptides are peptides that are generated by cleavage of the amyloid β precursor protein (APP) and can be found in the brains of Alzheimer's disease patients. Examples of amyloid β peptides include Aβ40 and Aβ42.

[0022] Amyloid A is a protein with a molecular weight of about 9 kDa that can form amyloid fibrils and deposit in various organs throughout the body. Amyloid A is generated by fibrillation of serum amyloid A protein (also called "SAA protein") with a molecular weight of about 12 to 14 kDa. SAA protein has isotypes such as SAA1 protein, SAA2 protein, and SAA4 protein, which are known to be mainly produced in the liver. In the present specification, the origin of amyloid A is not important, and amyloid A may be derived from any of the above SAA proteins.

[0023] "Amyloidosis" is a disease in which amyloid accumulates locally or throughout the body, causing damage to various organs such as the brain. Amyloidosis is a progressive, intractable disease in humans and animals, and 36 types are known depending on the type of causative protein. Amyloidosis can be classified into systemic amyloidosis, in which amyloid is deposited in various organs throughout the body, and localized amyloidosis, in which amyloid is deposited locally in certain organs. Examples of systemic amyloidosis include familial amyloid polyneuropathy (FAP), senile systemic amyloidosis (SSA), amyloid A amyloidosis (AA amyloidosis), ApoAII amyloidosis, transthyretin amyloidosis, fibrinogen amyloidosis, and dialysis amyloidosis. Examples of localized amyloidosis include Alzheimer's disease, cerebral amyloid angiopathy, AL amyloidosis, ApoAI amyloidosis, islet amyloidosis, insulin-derived amyloidosis, ACas-associated amyloidosis, amyloidogenic thyroid C-cell tumors, amyloidogenic odontogenic tumors, Parkinson's disease, Huntington's disease, and prion diseases.

[0024] Amyloid A amyloidosis (AA amyloidosis) is a disease caused by the aggregation of amyloid A, which becomes amyloid fibrils and is deposited in various organs throughout the body. In cattle, it is sometimes called "bovine amyloidosis." In cattle with AA amyloidosis, pathological examination reveals extensive amyloid deposition in various organs throughout the body, especially in the kidneys and liver. In humans, AA amyloidosis is often associated with autoimmune diseases, chronic infections, chronic inflammatory diseases, cancer, etc.

[0025] As used herein, the term "living body" refers to a cell (including cultured cells), tissue, organ, or individual. Examples of such an individual, but not limited to, include humans, livestock animals (cows, horses, sheep, goats, pigs, chickens, ostriches, etc.), race horses, pet animals (dogs, cats, rabbits, etc.), laboratory animals (mice, rats, guinea pigs, monkeys, marmosets, etc.), and wild and zoo animals (chimpanzees, wild cats, squirrels, eagles, flamingos, etc.), or cells, tissues, organs, or organs derived therefrom.

[0026] As used herein, the term "biological sample" refers to any sample derived from a living organism. Examples include tissues, cells, and body fluids. Examples of tissues include tissue homogenates, tissue sections, and biopsy tissues. The tissues and cells include, for example, tissues and cells of a subject that are affected or may be affected by a disease, as well as corresponding tissues and cells in a healthy subject, and may be tissues or cells derived from, for example, the brain, liver, spleen, heart, pancreas (including pancreatic islets), lungs, esophagus, kidneys, ovaries, stomach, large intestine, prostate, breast (including mammary glands), muscle, thyroid, and gums. Examples of body fluids include cerebrospinal fluid, interstitial fluid, blood (including serum, plasma, and interstitial fluid), lymph, extracts of each tissue or cell, pleural effusion, sputum, tears, nasal discharge, saliva, urine, and the like. Blood may be serum or plasma prepared from blood.

[0027] In the present specification, the term "subject" refers to a living body to which the detection method or disease differentiation support method of the present invention is applied. Examples of such a body include humans (particularly referred to as "subject" in this case), livestock animals (cattle, horses, sheep, goats, pigs, chickens, ducks, ostriches, etc.), racehorses, pet animals (dogs, cats, rabbits, etc.), laboratory animals (mice, rats, guinea pigs, monkeys, marmosets, etc.), wild and zoo animals (chimpanzees, wildcats, squirrels, eagles, flamingos, etc.), etc. For example, such a body includes humans, cattle, and other living bodies that have amyloidosis or may develop amyloidosis.

[0028] As used herein, the term "healthy individual" refers to an individual not suffering from a specific disease, preferably an individual not suffering from any disease. However, as used herein, healthy tissue is also included in the term "healthy individual" in the broad sense. Therefore, not only at the individual level, but also at the tissue level, such as a normal portion of a tissue taken from a patient, if the tissue is in a healthy state, it is called a healthy individual.

[0029] As used herein, the term "control" refers to a healthy individual, i.e., an individual not suffering from a particular disease (e.g., amyloidosis), preferably an individual not suffering from any disease. The control may be one individual or multiple individuals.

[0030] The term "detection" as used herein may be interchangeably referred to as inspection, measurement, determination, or determination assistance.

[0031] As used herein, "fluorescence fingerprint analysis" refers to an analysis method that includes irradiating a sample with excitation light while changing the wavelength of the excitation light stepwise or continuously, and measuring the fluorescence intensity while changing the wavelength of the fluorescence to be measured stepwise or continuously. As a result of the fluorescence fingerprint analysis, three-dimensional information consisting of the excitation wavelength, the fluorescence wavelength, and the fluorescence intensity can be obtained. In the fluorescence fingerprint analysis, it is possible to detect components such as misfolded proteins contained in a biological sample based on this three-dimensional information. The three-dimensional information obtained by the fluorescence fingerprint analysis is often expressed three-dimensionally by a contour plot, but the method of expressing the three-dimensional information is not particularly limited in this specification.

[0032] As used herein, "autofluorescence" refers to fluorescence emitted from a sample without relying on artificially added fluorescent substances, for example, fluorescence emitted from unlabeled endogenous substances in a living body or a biological sample. The autofluorescence detected by the method of the present invention is autofluorescence emitted from misfolded proteins in a biological sample. The autofluorescence detected by the method of the present invention is preferably autofluorescence that increases in misfolded proteins compared to correctly folded proteins, and more preferably autofluorescence specific to misfolded proteins. In the case of amyloid, it is preferably autofluorescence that increases in amyloid compared to amyloid precursor proteins, and may be autofluorescence based on a molecular structure specific to amyloid, for example, a cross-β sheet structure.

[0033] It has been found in the examples of this specification that at least two types of autofluorescence peaks can be detected in the fluorescence fingerprint analysis of a biological sample containing amyloid. In this specification, of the two types of autofluorescence peaks that can be found in the fluorescence fingerprint analysis of a biological sample containing amyloid, the peak with an excitation wavelength of less than 400 nm is referred to as the "amyloid first peak" and the peak with an excitation wavelength of 400 nm or more is referred to as the "amyloid second peak". The amyloid first peak and the amyloid second peak were first found in the bovine liver positive for AA amyloid in this example, but common peaks were also observed in similar wavelength ranges for other amyloids. Therefore, in this specification, peaks observed in similar wavelength ranges in a biological sample containing amyloid are referred to as the amyloid first peak and the amyloid second peak, regardless of the type of amyloid. In a biological sample containing amyloid, either the amyloid first peak or the amyloid second peak, or both, can be observed.

[0034] 1-3. Method The specific configurations of each step of the misfolded protein detection method of this embodiment differ depending on whether the biological sample is irradiated with excitation light having a specific excitation wavelength or the biological sample is irradiated with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range. Therefore, the two cases will be described below.

[0035] 1-3-1. When irradiating a biological sample with excitation light having a specific excitation wavelength When a biological sample is irradiated with excitation light having a specific excitation wavelength, the detection method of this embodiment includes an irradiation step and a detection step as essential steps.

[0036] (irradiation process) In this embodiment, the irradiating step is a step of irradiating the biological sample with excitation light having a specific excitation wavelength.

[0037] The excitation wavelength of the excitation light irradiated to the biological sample in this step is a wavelength capable of exciting misfolded proteins such as amyloid in the biological sample, for example, an excitation wavelength corresponding to amyloid peak 1 and / or amyloid peak 2. The excitation wavelength corresponding to amyloid peak 1 is about 350 nm, for example, 300 nm to 400 nm, 305 nm to 395 nm, 310 nm to 390 nm, 315 nm to 385 nm, 320 nm to 380 nm, 325 nm to 375 nm, 330 nm to 370 nm, 335 nm to 365 nm, 340 nm to 360 nm, or 345 nm to 355 nm, for example, it may be 350 nm. The excitation wavelength corresponding to the second amyloid peak is about 450 nm, and may be, for example, 400 nm to 500 nm, 405 nm to 495 nm, 410 nm to 490 nm, 415 nm to 485 nm, 420 nm to 480 nm, 425 nm to 475 nm, 430 nm to 470 nm, 435 nm to 465 nm, 440 nm to 460 nm, or 445 nm to 455 nm, for example, 450 nm.

[0038] In one embodiment, the excitation wavelength is 320 nm to 380 nm and / or 420 nm to 480 nm, for example, 325 nm to 375 nm and / or 425 nm to 475 nm; 330 nm to 370 nm and / or 430 nm to 470 nm; 335 nm to 365 nm and / or 435 nm to 465 nm; 340 nm to 360 nm and / or 440 nm to 460 nm; or 345 nm to 355 nm and / or 445 nm to 455 nm, for example, 350 nm and / or 450 nm.

[0039] The type of light source used for the excitation light irradiation in this step is not particularly limited as long as it is a light source capable of emitting excitation light having the above-mentioned wavelength. The specific configuration of the light source and auxiliary members such as a filter, a reflector, and / or a dichroic mirror that may be used in combination with the light source to select a specific excitation wavelength are described in detail in the third embodiment, and therefore will not be described here.

[0040] (Detection process) The detection step is a step of detecting autofluorescence emitted from the misfolded protein upon irradiation with excitation light.

[0041] The fluorescence wavelength of the autofluorescence detected in this step is the fluorescence wavelength of the fluorescence emitted from misfolded proteins such as amyloid in the biological sample, and is, for example, the fluorescence wavelength corresponding to the amyloid first peak and / or the amyloid second peak. The fluorescence wavelength corresponding to the first amyloid peak is about 430 nm, and may be, for example, 380 nm to 550 nm, 380 nm to 540 nm, 380 nm to 530 nm, 380 nm to 520 nm, 380 nm to 510 nm, 380 nm to 500 nm, 380 nm to 490 nm, 380 nm to 480 nm, 385 nm to 475 nm, 390 nm to 470 nm, 395 nm to 465 nm, 400 nm to 460 nm, 405 nm to 455 nm, 410 nm to 450 nm, 415 nm to 445 nm, 420 nm to 440 nm, or 425 nm to 435 nm, for example, 430 nm. The fluorescence wavelength corresponding to the second amyloid peak is about 480 nm, and may be, for example, 430 nm to 530 nm, 440 nm to 520 nm, 445 nm to 515 nm, 450 nm to 510 nm, 455 nm to 505 nm, 460 nm to 500 nm, 465 nm to 495 nm, 470 nm to 490 nm, or 475 nm to 485 nm, for example, 480 nm.

[0042] In one embodiment, the fluorescence wavelength of the autofluorescence detected in this step is 400 nm to 510 nm, and may be, for example, 405 nm to 505 nm; 410 nm to 450 nm and / or 460 nm to 500 nm; 415 nm to 445 nm and / or 465 nm to 495 nm; 420 nm to 440 nm and / or 470 nm to 490 nm; or 425 nm to 435 nm and / or 475 nm to 485 nm, for example, 430 nm and / or 480 nm.

[0043] In this step, the method for detecting the autofluorescence may be a measurement method known in the art. The detection of the autofluorescence can be performed by selecting the desired fluorescence wavelength using a filter, a reflector, and / or a dichroic mirror, etc., and then perceiving the visible light with the naked eye. For example, the detection may be performed by epifluorescence measurement. This method is a method in which excitation light is irradiated from above the sample to be measured, and the fluorescence emitted from the sample is measured by epifluorescence. In addition, the fluorescence emitted from a biological sample irradiated with an LED light source such as an LED handy light for fluorescence excitation can be perceived with the naked eye through observation goggles. Alternatively, the autofluorescence can be detected using a detection unit or detector described in the third aspect described later, for example, using a spectrophotometer such as a fluorometer.

[0044] 1-3-2. When irradiating a biological sample with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range When a biological sample is irradiated with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range, the detection method of this embodiment includes, as essential steps, an irradiation step, a measurement step, and a determination step.

[0045] (irradiation process) In this embodiment, the irradiation step is a step of irradiating the biological sample with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range.

[0046] In this specification, "irradiating a biological sample with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range" refers to irradiating a biological sample with excitation wavelengths at wavelength intervals that allow peaks corresponding to misfolded proteins such as amyloid in the biological sample (e.g., amyloid first peak and / or amyloid second peak) to be sufficiently identified. The wavelength intervals at which the excitation wavelength is changed may or may not be constant, and are not limited, but may be, for example, wavelength intervals of 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, 5 nm or less, 2 nm or less, or 1 nm or less.

[0047] The excitation wavelength range of the excitation light irradiated to the biological sample in this step is a wavelength range including wavelengths capable of exciting misfolded proteins such as amyloid in the biological sample, for example, a wavelength range including excitation wavelengths corresponding to amyloid first peak and / or amyloid second peak. The wavelength range including the excitation wavelength corresponding to amyloid first peak includes 350 nm, and may include, for example, 345 nm to 355 nm, 340 nm to 360 nm, 335 nm to 365 nm, 330 nm to 370 nm, 325 nm to 375 nm, 320 nm to 380 nm, 315 nm to 385 nm, 310 nm to 390 nm, 305 nm to 395 nm, or 300 nm to 400 nm. The wavelength range including the excitation wavelength corresponding to the second amyloid peak includes 450 nm, and may include, for example, 445 nm to 455 nm, 440 nm to 460 nm, 435 nm to 465 nm, 430 nm to 470 nm, 425 nm to 475 nm, 420 nm to 480 nm, 415 nm to 485 nm, 410 nm to 490 nm, 405 nm to 495 nm, or 400 nm to 500 nm.

[0048] In one embodiment, the excitation wavelength range of the excitation light irradiated to the biological sample includes 350 nm and / or 450 nm, and may include, for example, 345 nm to 355 nm and / or 445 nm to 455 nm; 340 nm to 360 nm and / or 440 nm to 460 nm; 335 nm to 365 nm and / or 435 nm to 465 nm; 330 nm to 370 nm and / or 430 nm to 470 nm; 325 nm to 375 nm and / or 425 nm to 475 nm; 320 nm to 380 nm and / or 420 nm to 480 nm; 315 nm to 385 nm and / or 415 nm to 485 nm; 310 nm to 390 nm and / or 410 nm to 490 nm; 305 nm to 395 nm and / or 405 nm to 495 nm; or 300 nm to 500 nm.

[0049] The light source used in this step and the spectroscopic method or spectrometer for changing the excitation wavelength stepwise or continuously are not particularly limited in type as long as they can emit excitation light while changing the wavelength stepwise or continuously within the above-mentioned excitation wavelength range. The specific configurations of the light source and spectrometer are described in detail in the third embodiment, and therefore will not be described here.

[0050] (Measurement process) The measurement step is a step of measuring the wavelength and intensity of the autofluorescence emitted from the biological sample by irradiation with excitation light. More specifically, in this step, the fluorescence intensity can be measured while changing the fluorescence wavelength of the measurement target stepwise or continuously.

[0051] In this specification, "measuring the fluorescence intensity while changing the fluorescence wavelength of the measurement target stepwise or continuously" means measuring the fluorescence intensity while changing the observed fluorescence wavelength at wavelength intervals that allow sufficient identification of peaks corresponding to misfolded proteins such as amyloid in a biological sample (e.g., amyloid first peak and / or amyloid second peak). The wavelength intervals at which the fluorescence wavelength is changed may or may not be constant, and are not limited, but may be, for example, wavelength intervals of 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, 5 nm or less, 2 nm or less, or 1 nm or less.

[0052] The fluorescence wavelength range of the autofluorescence detected in this step is a wavelength range that includes the fluorescence wavelengths emitted from misfolded proteins such as amyloid in a biological sample, for example, a wavelength range that includes the fluorescence wavelengths corresponding to the amyloid first peak and / or the amyloid second peak. The fluorescence wavelength range including the fluorescence wavelength corresponding to the first amyloid peak includes 430 nm, and may include, for example, 425 nm to 435 nm, 420 nm to 440 nm, 415 nm to 445 nm, 410 nm to 450 nm, 405 nm to 455 nm, 400 nm to 460 nm, 395 nm to 465 nm, 390 nm to 470 nm, 385 nm to 475 nm, 380 nm to 480 nm, 380 nm to 490 nm, 380 nm to 500 nm, 380 nm to 510 nm, 380 nm to 520 nm, 380 nm to 530 nm, 380 nm to 540 nm, or 380 nm to 550 nm. The fluorescence wavelength range including the fluorescence wavelength corresponding to the second amyloid peak includes 480 nm, and may include, for example, 475 nm to 485 nm, 470 nm to 490 nm, 465 nm to 495 nm, 460 nm to 500 nm, 455 nm to 505 nm, 450 nm to 510 nm, 445 nm to 515 nm, 440 nm to 520 nm, 435 nm to 525 nm, or 430 nm to 530 nm.

[0053] In one embodiment, the fluorescence wavelength range of the autofluorescence to be detected includes 430 nm and / or 480 nm, and may include, for example, 425 nm to 435 nm and / or 475 nm to 485 nm; 420 nm to 440 nm and / or 470 nm to 490 nm; 415 nm to 445 nm and / or 465 nm to 495 nm; 410 nm to 450 nm and / or 460 nm to 500 nm; 405 nm to 515 nm; or 400 nm to 510 nm.

[0054] In this step, the spectroscopic method or spectrometer and the detection method or detector for measuring the autofluorescence while changing the fluorescence wavelength of the measurement target stepwise or continuously are not limited. The specific configurations of the spectrometer and detector are described in detail in the third embodiment, and therefore will not be described here. For example, a spectrophotometer such as a fluorometer may be used.

[0055] This step provides measurements of the wavelength and intensity of the autofluorescence emitted from the biological sample. The measurement data obtained in this step may include three-dimensional information consisting of the excitation wavelength, the fluorescence wavelength, and the fluorescence intensity.

[0056] (Judgment process) The determination step is a step of determining the presence or absence of a misfolded protein such as amyloid that emits autofluorescence based on the measured value obtained in the measurement step. In this step, it can be determined that the biological sample contains a misfolded protein when the measured value obtained in the measurement step includes an autofluorescence peak of the misfolded protein to be detected.

[0057] In one embodiment, the misfolded protein is amyloid, and a biological sample can be determined to contain amyloid in the following cases (A) and / or (B): (A) Irradiation of a biological sample with excitation light in an excitation wavelength range including 300 nm to 400 nm, 305 nm to 395 nm, 310 nm to 390 nm, 315 nm to 385 nm, or 320 nm to 380 nm (e.g., irradiation of excitation light in an excitation wavelength range including 325 nm to 375 nm, 330 nm to 370 nm, 335 nm to 365 nm, 340 nm to 360 nm, or 345 nm to 355 nm). and / or when the peak wavelength of the autofluorescence emitted from the material is within the range of 380 nm to 480 nm, 385 nm to 475 nm, 390 nm to 470 nm, or 395 nm to 465 nm, preferably 400 nm to 460 nm (for example, 405 nm to 455 nm, 410 nm to 450 nm, 415 nm to 445 nm, 420 nm to 440 nm, or 425 nm to 435 nm). (B) When the peak wavelength of the autofluorescence emitted from a biological sample by irradiation with excitation light in an excitation wavelength range including 400 nm to 500 nm, 405 nm to 495 nm, 410 nm to 490 nm, 415 nm to 485 nm, or 420 nm to 480 nm (for example, irradiation with excitation light in an excitation wavelength range including 425 nm to 475 nm, 430 nm to 470 nm, 435 nm to 465 nm, 440 nm to 460 nm, or 445 nm to 455 nm) is within the range of 430 nm to 530 nm, 435 nm to 525 nm, 440 nm to 520 nm, or 445 nm to 515 nm, preferably 450 nm to 510 nm (for example, 455 nm to 505 nm, 460 nm to 500 nm, 465 nm to 495 nm, 470 nm to 490 nm, or 475 nm to 485 nm).

[0058] In a further embodiment, the misfolded protein is amyloid, and the biological sample can be determined to contain amyloid in the following cases (a) and / or (b): (a) the peak wavelength of the autofluorescence emitted from the biological sample when irradiated with excitation light having an excitation wavelength range of 320 nm to 380 nm is in the range of 400 nm to 460 nm, and / or (b) When the peak wavelength of the autofluorescence emitted from a biological sample by irradiation with excitation light in the excitation wavelength range including 420 nm to 480 nm is in the range of 450 nm to 510 nm.

[0059] 1-4.Effects According to the misfolded protein detection method of this embodiment, amyloids such as amyloid β and amyloid A and misfolded proteins can be rapidly and easily detected in a biological sample such as tissue (for example, tissue extracted by biopsy or autopsy from a human or animal, or tissue extracted from a livestock animal such as a cow) or body fluid without labeling. Furthermore, the method of the present invention also provides a meat inspection method that can rapidly and easily detect amyloid in a biological sample without labeling.

[0060] The misfolded protein detection method of this embodiment enables rapid detection of amyloid without labeling, as compared with histopathological examination methods and positron emission tomography (e.g., amyloid PET) that have been conventionally used for detecting amyloid. Furthermore, the detection method of this embodiment has the advantage that it can be carried out extremely easily and simply, and homogenized tissue samples can also be used, and therefore does not require proficiency in histopathology for carrying it out.

[0061] 2. Methods to assist in the diagnosis of amyloidosis 2-1. Overview A second aspect of the present invention is a method for assisting in the diagnosis of amyloidosis. The disease diagnosis assistance method of this embodiment includes an irradiation step, a measurement step, and a determination step. According to the disease diagnosis assistance method of this embodiment, it is possible to assist in the diagnosis of amyloidosis in a subject quickly and easily without labeling.

[0062] 2-2. Method The amyloidosis diagnosis assistance method of this embodiment includes an irradiation step, a measurement step, and a judgment step as essential steps. The specific configuration of each step in the method of this embodiment differs depending on whether the biological sample is irradiated with excitation light having a specific excitation wavelength or the biological sample is irradiated with excitation light while the wavelength of the excitation light is changed stepwise or continuously within a specific excitation wavelength range. Therefore, the following description will be divided into two cases.

[0063] 2-2-1. When irradiating a biological sample with excitation light having a specific excitation wavelength (irradiation process) In the case where a biological sample is irradiated with excitation light having a specific excitation wavelength, the irradiation step is a step of irradiating biological samples derived from a subject and a control with excitation light having a specific wavelength. This step is similar to the irradiation step in "1-3-1. In the case where a biological sample is irradiated with excitation light having a specific excitation wavelength" except that at least two biological samples derived from a subject and a control are used.

[0064] (Measurement process) When the biological sample is irradiated with excitation light having a specific excitation wavelength, the measurement step is a step of measuring the fluorescence intensity of the autofluorescence emitted from the amyloid in the biological sample by irradiation with the excitation light. In this step, the fluorescence intensity of the autofluorescence can be measured in accordance with the detection step in "1-3-1. When the biological sample is irradiated with excitation light having a specific excitation wavelength", except that at least two biological samples derived from a subject and a control are used. This step provides the fluorescence intensity of the test subject and the fluorescence intensity of the control subject.

[0065] (Judgment process) When excitation light having a specific excitation wavelength is irradiated onto a biological sample, the determination step is a step of comparing the fluorescence intensities obtained in the measurement step between the subject and the control, and determining that the subject is suffering from amyloidosis if the fluorescence intensity of the subject is higher than that of the control.

[0066] In this step, the method of comparing the fluorescence intensity of the subject and the control is not limited. For example, a cutoff value for the fluorescence intensity of the subject is determined based on the fluorescence intensity of the control, and the comparison is based on the cutoff value. That is, a predetermined value is determined as the cutoff value, and if the measured value is equal to or greater than the cutoff value, it can be determined that the fluorescence intensity of the subject is greater than the fluorescence intensity of the control.

[0067] The cutoff value refers to a boundary value for comparing the fluorescence intensity of a subject and a control. The cutoff value can usually be calculated based on the disease incidence rate and the sensitivity and specificity calculated from a receiver operating characteristic curve (ROC curve). There is no particular limitation on the method for setting the cutoff value.

[0068] For example, the fluorescence intensity of a healthy subject not suffering from amyloidosis or the average fluorescence intensity in a group of healthy subjects can be set as a cutoff value, and when the fluorescence intensity of the subject is higher than the cutoff value, it can be determined that the fluorescence intensity of the subject is greater than the fluorescence intensity of the control subject.

[0069] Alternatively, a cutoff value may be set to 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 6 times or more the fluorescence intensity of a healthy subject not suffering from amyloidosis or the average fluorescence intensity in a group of healthy subjects, and when the fluorescence intensity of the subject is higher than the cutoff value, it may be determined that the fluorescence intensity of the subject is higher than the fluorescence intensity of the control subject.

[0070] Alternatively, the measured values ​​obtained from the control group may be classified by percentile, and the percentile value used for the classification may be used as the cutoff value. For example, the 95th percentile of the fluorescence intensity obtained from the control may be used as the cutoff value, and if the fluorescence intensity of the test subject is equal to or greater than the 95th percentile, it may be determined that the fluorescence intensity of the test subject is greater than that of the control.

[0071] The fluorescence intensity of the control healthy subject is different from that of the subject, and does not necessarily need to be measured each time. For example, if the amount of sample used in the measurement, the measurement method, and the measurement conditions are kept constant, the fluorescence intensity of the control healthy subject previously measured can be reused.

[0072] 2-2-2. When irradiating a biological sample with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range (irradiation process) In the case where the biological sample is irradiated with excitation light while the wavelength of the excitation light is changed stepwise or continuously within a specific excitation wavelength range, the irradiation step is a step of irradiating the biological sample derived from the subject with the excitation light while the wavelength of the excitation light is changed stepwise or continuously within a specific excitation wavelength range. This step is similar to the irradiation step in "1-3-2. In the case where the biological sample is irradiated with excitation light while the wavelength of the excitation light is changed stepwise or continuously within a specific excitation wavelength range".

[0073] (Measurement process) In the case where the biological sample is irradiated with excitation light while the wavelength of the excitation light is changed stepwise or continuously within a specific excitation wavelength range, the measurement step is a step of measuring the wavelength and intensity of the autofluorescence emitted from the biological sample by irradiation with the excitation light. This step is similar to the measurement step in "1-3-2. In the case where the biological sample is irradiated with excitation light while the wavelength of the excitation light is changed stepwise or continuously within a specific excitation wavelength range."

[0074] This step provides measurements of the wavelength and intensity of the autofluorescence emitted by the biological sample. The measurement data obtained in this step may include three-dimensional information consisting of the excitation wavelength, the fluorescence wavelength, and the fluorescence intensity. For example, a contour plot may be created from the three-dimensional information obtained in this step.

[0075] (Judgment process) In the case where the excitation light is irradiated onto the biological sample while the wavelength of the excitation light is changed stepwise or continuously within a specific excitation wavelength range, the determination step is a step of determining that the subject suffers from amyloidosis when the measured value obtained in the measurement step includes a peak of autofluorescence of amyloid. Specifically, the subject can be determined to suffer from amyloidosis in the case of (A) and / or (B) or (a) and / or (b) described in the determination step in "1-3-2. In the case where the excitation light is irradiated onto the biological sample while the wavelength of the excitation light is changed stepwise or continuously within a specific excitation wavelength range."

[0076] 2-3.Effects According to the present embodiment of the method for assisting in diagnosis of amyloidosis, amyloidosis can be diagnosed quickly and easily without labeling.

[0077] 3. Misfolded protein detection device A third aspect of the present invention is an apparatus for detecting misfolded proteins. The misfolded protein detection device of this embodiment includes a light source and an optical unit as essential components, and may further include a detection unit, a wavelength varying unit, an observation stand, a camera, hardware, and / or software as optional components.

[0078] 3-1.Configuration In one embodiment, the misfolded protein detection device of this aspect includes a light source and an optical unit.

[0079] In a further embodiment, the misfolded protein detection device of this embodiment includes a light source, an optical unit, and a detection unit, and optionally includes a wavelength variable unit. Hereinafter, each of the components of the light source, the optical unit, the detection unit, and the wavelength variable unit in the misfolded protein detection device of this embodiment will be specifically described.

[0080] (light source) The light source is a light source that emits excitation light of a wavelength capable of exciting misfolded proteins such as amyloid in a biological sample. A light source that emits excitation light of an excitation wavelength capable of exciting the autofluorescence of a misfolded protein, for example, an excitation wavelength corresponding to the first amyloid peak and / or the second amyloid peak, or a light source whose irradiation wavelength range includes an excitation wavelength corresponding to the first amyloid peak and / or the second amyloid peak, can be used.

[0081] In one embodiment, the specific excitation wavelength of the excitation light emitted from the light source may be about 350 nm, for example, 300 nm to 400 nm, 305 nm to 395 nm, 310 nm to 390 nm, 315 nm to 385 nm, 320 nm to 380 nm, 325 nm to 375 nm, 330 nm to 370 nm, 335 nm to 365 nm, 340 nm to 360 nm, or 345 nm to 355 nm; or about 450 nm, for example, 400 nm to 500 nm, 405 nm to 495 nm, 410 nm to 490 nm, 415 nm to 485 nm, 420 nm to 480 nm, 425 nm to 475 nm, 430 nm to 470 nm, 435 nm to 465 nm, 440 nm to 460 nm, or 445 nm to 455 nm.

[0082] In further embodiments, the specific excitation wavelengths of the excitation light emitted from the light source are 320 nm to 380 nm and / or 420 nm to 480 nm, and may be, for example, 325 nm to 375 nm and / or 425 nm to 475 nm; 330 nm to 370 nm and / or 430 nm to 470 nm; 335 nm to 365 nm and / or 435 nm to 465 nm; 340 nm to 360 nm and / or 440 nm to 460 nm; 345 nm to 355 nm and / or 445 nm to 455 nm; or 350 nm and / or 450 nm.

[0083] In another embodiment, the specific excitation wavelength range of the excitation light emitted from the light source may include about 350 nm, e.g., 300 nm to 400 nm, 305 nm to 395 nm, 310 nm to 390 nm, 315 nm to 385 nm, 320 nm to 380 nm, 325 nm to 375 nm, 330 nm to 370 nm, 335 nm to 365 nm, 340 nm to 360 nm, or 345 nm to 355 nm; and / or about 450 nm, e.g., 400 nm to 500 nm, 405 nm to 495 nm, 410 nm to 490 nm, 415 nm to 485 nm, 420 nm to 480 nm, 425 nm to 475 nm, 430 nm to 470 nm, 435 nm to 465 nm, 440 nm to 460 nm, or 445 nm to 455 nm.

[0084] In further embodiments, specific excitation wavelength ranges of the excitation light emitted from the light source can include 320 nm to 380 nm and / or 420 nm to 480 nm, for example, 325 nm to 375 nm and / or 425 nm to 475 nm; 330 nm to 370 nm and / or 430 nm to 470 nm; 335 nm to 365 nm and / or 435 nm to 465 nm; 340 nm to 360 nm and / or 440 nm to 460 nm; 345 nm to 355 nm and / or 445 nm to 455 nm; or 350 nm and / or 450 nm.

[0085] The specific type of light source is not limited as long as it can irradiate excitation light in the above wavelength range. Examples include black light, LED light sources such as UV-LED (ultraviolet light emitting diode) and visible light LED, deuterium lamp, xenon lamp, tungsten lamp, mercury lamp, metal halide lamp, halogen lamp, incandescent lamp, fluorescent lamp, and wavelength tunable laser. However, when a full-wavelength light source is used as an excitation light source, auxiliary members such as a filter (an excitation filter that transmits only excitation light having a specific wavelength among the light emitted from the light source, for example, a bandpass filter) for selecting only light in a required wavelength range and a reflector (a member having a mirror surface that can reflect all or part of light; for example, a reflecting mirror that changes the direction of light emitted from the light source and a dichroic mirror that reflects only excitation light having a specific wavelength and transmits light of other wavelengths) may be required.

[0086] (Optical unit) The optical unit is an optical unit capable of separating the fluorescence emitted from the biological sample from the excitation light based on the difference in wavelength. Specifically, the optical unit can separate the fluorescence emitted from misfolded proteins such as amyloid in the biological sample. For example, the optical unit can separate the fluorescence wavelengths corresponding to the amyloid first peak and / or the amyloid second peak.

[0087] In one embodiment, the fluorescence wavelength of the fluorescence separated by the optical unit is about 430 nm, for example, 380 nm to 550 nm, 380 nm to 540 nm, 380 nm to 530 nm, 380 nm to 520 nm, 380 nm to 510 nm, 380 nm to 500 nm, 380 nm to 490 nm, 380 nm to 480 nm, 385 nm to 475 nm, 390 nm to 470 nm, 395 nm to 465 nm, 400 nm to 460 nm, 405 nm to 475 nm, 405 nm to 485 nm, 405 nm to 490 nm, 405 nm to 475 nm, 405 nm to 470 nm, 405 nm to 470 nm, 405 nm to 470 nm, 405 nm to 485 nm, 405 nm to 49 ... or about 480 nm, for example, 430 nm to 530 nm, 440 nm to 520 nm, 445 nm to 515 nm, 450 nm to 510 nm, 455 nm to 505 nm, 460 nm to 500 nm, 465 nm to 495 nm, 470 nm to 490 nm, 475 nm to 485 nm, or 480 nm.

[0088] In a further embodiment, the fluorescence wavelength of the fluorescence separated by the optical unit is 400 nm to 510 nm, and may be, for example, 405 nm to 505 nm; 410 nm to 450 nm and / or 460 nm to 500 nm; 415 nm to 445 nm and / or 465 nm to 495 nm; 420 nm to 440 nm and / or 470 nm to 490 nm; 425 nm to 435 nm and / or 475 nm to 485 nm; or 430 nm and / or 480 nm.

[0089] In another embodiment, the fluorescence wavelength range of the fluorescence separated by the optical unit is about 430 nm, for example, 380 nm to 550 nm, 380 nm to 540 nm, 380 nm to 530 nm, 380 nm to 520 nm, 380 nm to 510 nm, 380 nm to 500 nm, 380 nm to 490 nm, 380 nm to 480 nm, 385 nm to 475 nm, 390 nm to 470 nm, 395 nm to 465 nm, 400 nm to 460 nm, 405 nm to 455 nm, m, 410 nm to 450 nm, 415 nm to 445 nm, 420 nm to 440 nm, 425 nm to 435 nm, or 430 nm; and / or about 480 nm, for example, 430 nm to 530 nm, 440 nm to 520 nm, 445 nm to 515 nm, 450 nm to 510 nm, 455 nm to 505 nm, 460 nm to 500 nm, 465 nm to 495 nm, 470 nm to 490 nm, 475 nm to 485 nm, or 480 nm.

[0090] In a further embodiment, the fluorescence wavelength range of the fluorescence separated by the optical unit may include 400 nm to 510 nm, for example, 405 nm to 505 nm; 410 nm to 450 nm and / or 460 nm to 500 nm; 415 nm to 445 nm and / or 465 nm to 495 nm; 420 nm to 440 nm and / or 470 nm to 490 nm; 425 nm to 435 nm and / or 475 nm to 485 nm; or 430 nm and / or 480 nm. It is preferable that the fluorescence wavelength range of the fluorescence separated by the optical unit does not include the excitation wavelength range of the excitation light.

[0091] The specific type of optical unit is not limited as long as it can separate the fluorescence in the above wavelength range. The optical unit may include a filter (fluorescence filter) for selecting only the fluorescence in the required wavelength range, a reflector (a reflecting mirror or a dichroic mirror), a slit, or the like as necessary.

[0092] (Detection unit) The detection unit is capable of detecting autofluorescence emitted from a biological sample and measuring the intensity of the autofluorescence. The type of detector included in the detection unit is not limited, and may be, for example, an imaging element such as a CCD, a CMOS, or an organic thin-film imaging element, a photomultiplier tube (PMT), an InGaAs detector, a Si photodiode detector, or a PbS detector.

[0093] (Wavelength fluctuation part) The wavelength variation section is a wavelength variation section that changes the wavelength of the excitation light and / or fluorescence in a stepwise or continuous manner. The wavelength range of the excitation light that can be changed stepwise or continuously by the wavelength variation section can be the excitation wavelength described for the light source described above, or a range including that. For example, the wavelength of the excitation light can be changed in the entire wavelength range. Similarly, the fluorescence wavelength range of the fluorescence that can be changed stepwise or continuously by the wavelength variation section can be the fluorescence wavelength described for the optical unit described above, or a range including that. For example, the fluorescence wavelength can be changed in the entire wavelength range.

[0094] The wavelength varying unit includes a spectrometer that can separate the excitation light or the fluorescence according to wavelength. Examples of the spectrometer include a prism and a diffraction grating.

[0095] 3-2.Effects According to the misfolded protein detection device of this embodiment, misfolded proteins in biological samples can be detected quickly and easily in a small-scale laboratory. Unlike conventional histopathological examination methods, the device of this embodiment allows misfolded proteins to be detected easily and simply without labeling, without requiring mastery of histopathology. EXAMPLES

[0096] Example 1: Detection of amyloid in bovine liver by fluorescence fingerprint analysis (the purpose) Amyloid in bovine liver is detected by fluorescence fingerprint analysis.

[0097] (method) The livers extracted from four cows (all female Holsteins) aged 3 to 8 years were examined. Histopathological examination revealed that the livers of two of the four cows were negative for AA amyloid, while the livers of the remaining two cows were positive for AA amyloid. A sample (several tens of mg) obtained by trimming a part of fresh or frozen liver tissue was placed in about 2 to 4 mL of ultrapure water in a screw-cap tube together with zirconia beads (Nikkato, YTZ-3), and homogenized for 120 seconds at 2500 rpm under ice-cooled conditions in a bead crusher (Taitec, μT-12). The crushed sample was diluted about 100 to 1000 times with ultrapure water, and ultrasonicated for 5 minutes at 40 kHz using an ultrasonic crusher to obtain a well-dispersed sample (1 to 10 mg / mL). 1 mL of the unlabeled sample after ultrasonic crushing was transferred to an acrylic four-sided transmission disposable cell (Fisher scientific, 14-955-130), and a fluorescence fingerprint analysis was performed using a fluorescence spectrophotometer (JASCO, FP8300) under conditions of medium measurement sensitivity and excitation / fluorescence bandwidth of 5 nm, with an excitation wavelength band of 250 to 600 nm / fluorescence wavelength band of 250 to 600 nm.

[0098] (result) In the amyloid-negative liver homogenate, a major peak was detected in the excitation wavelength band of 260-310 nm / emission wavelength band of 290-360 nm, which corresponds to the absorption peak of aromatic amino acids (Figure 2). Similar results were obtained for another amyloid-negative case (one case).

[0099] On the other hand, in the amyloid-positive liver homogenate, in addition to the absorption peaks of aromatic amino acids, significant autofluorescence peaks were detected in the excitation wavelength range of 320nm to 380nm / fluorescence wavelength range of 400nm to 460nm and the excitation wavelength range of 420nm to 480nm / fluorescence wavelength range of 450nm to 510nm (Figure 3). Similar results were obtained for another amyloid-positive case (one case). Hereinafter, the peak observed in the excitation wavelength range of 320nm to 380nm / fluorescence wavelength range of 400nm to 460nm in the amyloid-positive liver homogenate is referred to as the "amyloid first peak", and the peak observed in the excitation wavelength range of 420nm to 480nm / fluorescence wavelength range of 450nm to 510nm is referred to as the "amyloid second peak".

[0100] Example 2: Detection of amyloid in mouse spleen and cat liver by fluorescence fingerprint analysis (the purpose) Amyloid in mouse spleens and cat livers affected by AA amyloidosis is detected by fluorescence fingerprint analysis.

[0101] (Methods and Results) Fluorescence fingerprint analysis was performed in the same manner as above on a spleen isolated from a mouse (C57BL / 6J strain, female, 8 weeks old) with AA amyloidosis and a liver isolated from a cat (mixed breed, female, 3 years old) with AA amyloidosis. Tissue homogenates were prepared from samples obtained by trimming portions from frozen mouse spleen and cat liver in the same manner as in Example 1, and fluorescence fingerprint analysis was performed using a fluorescence spectrophotometer.

[0102] Fluorescence fingerprint analysis showed that two peaks were observed in both mouse spleens and cat livers affected with AA amyloidosis, corresponding to the amyloid 1 and amyloid 2 peaks found in amyloid-positive bovine liver.

[0103] Example 3: Detection of other amyloids by fluorescence fingerprint analysis (the purpose) Other in vitro prepared amyloids are detected by fluorescence fingerprint analysis, including amyloid forms of LPS binding protein, Aβ42 peptide, and α-casein protein.

[0104] (method) According to the method described in the previous paper (Murakami T, et al., Amyloid 27(1): 25-35, 2020.), LPS-binding protein, Aβ42 peptide, and α-casein protein were cultured in a neutral solvent for one week, and amyloid was prepared from each protein. Specifically, synthetic peptides were used for LPS-binding protein and Aβ42 peptide, and E. coli recombinant protein was used for α-casein protein. Citrate buffer was used as a neutral solvent. The incubation was performed at 37 °C and a shaking speed of 800 rpm in 800 μL of solution containing each protein at 100 μM. After incubation, the samples were observed under an electron microscope, and it was observed that non-branched fibril-like fibers with a diameter of about 10 nm were formed in all samples, and that they had an amyloid morphology.

[0105] 1 mL of culture medium containing approximately 0.5 to 1 mg / mL of any of the above amyloid forms of protein was transferred to an acrylic four-sided disposable cell, and a fluorescence fingerprint analysis was performed using a fluorescence spectrophotometer under the same conditions as in Example 1, with an excitation wavelength band of 250 to 600 nm / fluorescence wavelength band of 250 to 600 nm.

[0106] The results are shown in Figures 4 to 6. In the amyloid form of LPS-binding protein, a significant autofluorescence peak was detected in the excitation wavelength range of 320 nm to 380 nm / fluorescence wavelength range of 390 nm to 480 nm (Figure 4). This peak was similar to the amyloid first peak found in the amyloid-positive bovine liver in Example 1.

[0107] In the amyloid form of Aβ42 peptide, a prominent autofluorescence peak was detected in the excitation wavelength range of 390 nm to 480 nm / fluorescence wavelength range of 450 nm to 530 nm (FIG. 5). This peak was similar to the amyloid second peak found in the amyloid-positive bovine liver in Example 1.

[0108] In addition, in the amyloid form of α-casein protein, a significant autofluorescence peak was detected in the excitation wavelength range of 420 nm to 460 nm / fluorescence wavelength range of 450 nm to 550 nm (FIG. 6). This peak was similar to the amyloid second peak found in the amyloid-positive bovine liver in Example 1.

Claims

1. 1. A method for detecting amyloid in a biological sample, comprising: Irradiating the biological sample with excitation light having a specific excitation wavelength; and detecting autofluorescence emitted from the amyloid upon irradiation with the excitation light Including, The above method, wherein the specific excitation wavelength is 330 nm to 370 nm and / or 420 nm to 480 nm, and the fluorescence wavelength of the autofluorescence is 400 nm to 510 nm.

2. 1. A method for detecting amyloid in a biological sample, comprising: Irradiating the biological sample with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range; measuring the wavelength and intensity of autofluorescence emitted from the biological sample upon irradiation with the excitation light; and A step of determining the presence or absence of amyloid that emits autofluorescence based on the obtained measurement value. Including, The method as described above, wherein in the determination step, the biological sample is determined to contain amyloid if the following (a) and / or (b) is met: (a) the peak wavelength of the autofluorescence emitted from the biological sample upon irradiation with excitation light having an excitation wavelength range including 330 nm to 370 nm is in the range of 400 nm to 460 nm; and / or (b) When irradiated with excitation light having a wavelength range of 420 nm to 480 nm, the peak wavelength of the autofluorescence emitted from the biological sample is in the range of 450 nm to 510 nm.

3. The method of claim 1 or 2, wherein the biological sample is a tissue or a body fluid.

4. The method of claim 3 , wherein the tissue is a tissue homogenate.

5. A method for assisting in the diagnosis of amyloidosis, comprising: Irradiating biological samples from non-human subjects and controls with excitation light having a specific excitation wavelength; measuring the fluorescence intensity of autofluorescence emitted from amyloid in the biological sample by irradiation with the excitation light; comparing the obtained fluorescence intensity between the subject and the control, and determining that the subject is affected by amyloidosis if the fluorescence intensity of the subject is higher than that of the control. Including, the specific excitation wavelength is 330 nm to 370 nm and / or 420 nm to 480 nm; The method as described above, wherein the fluorescence wavelength of the autofluorescence measured in the measuring step is 400 nm to 510 nm.

6. A method for assisting in the diagnosis of amyloidosis, comprising: A step of irradiating a biological sample derived from a subject other than a human with excitation light while changing the wavelength of the excitation light stepwise or continuously within a specific excitation wavelength range; measuring the wavelength and intensity of autofluorescence emitted from the biological sample upon irradiation with the excitation light; and determining, based on the obtained measured value, that the subject is affected with amyloidosis in the following cases (a) and / or (b): wherein the specific excitation wavelength ranges include 330 nm to 370 nm and / or 420 nm to 480 nm. (a) the peak wavelength of the autofluorescence emitted from the biological sample upon irradiation with excitation light having an excitation wavelength range including 330 nm to 370 nm is in the range of 400 nm to 460 nm; and / or (b) When irradiated with excitation light having a wavelength range of 420 nm to 480 nm, the peak wavelength of the autofluorescence emitted from the biological sample is in the range of 450 nm to 510 nm.

7. The method of claim 5 or 6, wherein the subject is a livestock animal.

8. The method according to any one of claims 5 to 7, wherein the amyloidosis is AA amyloidosis.

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