WO2020138265A1 - Procédé, appareil et programme pour évaluer la formation d'amyloïde - Google Patents

Procédé, appareil et programme pour évaluer la formation d'amyloïde Download PDF

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WO2020138265A1
WO2020138265A1 PCT/JP2019/051077 JP2019051077W WO2020138265A1 WO 2020138265 A1 WO2020138265 A1 WO 2020138265A1 JP 2019051077 W JP2019051077 W JP 2019051077W WO 2020138265 A1 WO2020138265 A1 WO 2020138265A1
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test substance
value
standard deviation
amyloid
calculated
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清孝 徳楽
幸司 上井
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国立大学法人室蘭工業大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to a method for evaluating amyloid formation using a fluorescent probe, and a device and a program used in the method.
  • Alzheimer's dementia is caused by the aggregation of amyloid ⁇ protein, which is a secretase degradation product of amyloid precursor protein, to form amyloid, which is then deposited in the brain.
  • Amyloid is a fibrillar supramolecular polymer produced by protein misfolding, and is known to be involved in many diseases such as Parkinson's disease, Huntington's disease and prion disease in addition to Alzheimer's disease. These diseases in which dysfunction is caused by amyloid deposits are called amyloidosis.
  • amyloid formation For the fundamental prevention and treatment of amyloidosis, the inhibition of amyloid formation, that is, the inhibition of polymerization and aggregation of amyloid-forming proteins such as amyloid ⁇ protein has attracted attention as a drug target.
  • the present inventors established a method for evaluating aggregation of amyloid ⁇ protein using a nanoprobe labeled with a quantum dot for amyloid ⁇ protein, and developed a trace high throughput screening system for determining amyloid formation inhibitory activity (non- Patent documents 1 and 2).
  • This method takes advantage of the fact that when quantum dot nanoprobes are incorporated into amyloid-forming protein aggregates, the density increases compared to when they were present in the free state, resulting in a bias in the fluorescence detection position. It was done.
  • SD standard deviation
  • Patent Document 1 discloses another quantum dot nanoprobe in which a peptide that forms an aggregate with an amyloidogenic protein is labeled with a quantum dot, and a plurality of types of amyloid can be obtained by using this probe in the above method. It has been described that aggregation of morphogenic proteins was detected.
  • the present invention aims to provide a means for highly accurately evaluating amyloid formation.
  • the present inventors in the evaluation method of amyloid formation using the quantum dot nanoprobe described above, the ratio of the total brightness value of each pixel to the total theoretical maximum brightness value of each pixel included in the image (hereinafter, It was found that the amyloid formation can be evaluated with high accuracy by adopting an exposure condition in which the Sum Intensity) is within a predetermined range. Furthermore, they have found that the accuracy of the evaluation system can be further improved and the sensitivity can be improved by performing a predetermined brightness value correction, and have completed the following inventions.
  • the agglutination reaction step the agglutination reaction is performed using multiple concentrations of the test substance, the fluorescence of the agglutination reaction product under each test substance concentration is imaged in the imaging step, and each test substance concentration is measured in the standard deviation calculation step. The corresponding luminance value standard deviation is calculated, and in addition to the determination of inhibitory activity in the activity determination step, the 50% effective concentration (EC 50 ) is determined from the inhibition curve showing the relationship between the concentration of the test substance and the luminance value standard deviation.
  • the method according to any one of 1) to (4).
  • amyloid-forming protein according to any one of (1) to (5), wherein the amyloid-forming protein is selected from the group consisting of amyloid ⁇ protein, tau and ⁇ -synuclein, partial peptides thereof and derivatives thereof.
  • the amyloidogenic protein is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 4, or any of the above three amino acid sequences
  • the method according to any one of (1) to (6) which is a peptide having an amino acid sequence having 90% or more sequence identity with the peptide and having an amyloid-forming ability.
  • the fluorescent probe capable of binding to amyloid formed by the polymerization of the amyloidogenic protein is a fluorescently labeled peptide having the ability to form amyloid with the amyloidogenic protein, The method according to any one of (1) to (7).
  • each of the amyloidogenic protein and the peptide in the fluorescent probe has the amino acid sequence represented by SEQ ID NO: 3.
  • the concentration of amyloidogenic protein at the start of the reaction in the agglutination reaction step is 10 ⁇ M, and the concentration of the fluorescent probe is 50 nM.
  • the fluorescent probe capable of binding to amyloid formed by the polymerization of the amyloid-forming protein is an amyloid-binding fluorescent dye or a fluorescently labeled anti-amyloid-specific antibody.
  • the method according to (13), wherein the amyloid-binding fluorescent dye is selected from the group consisting of Congo red, thioflavin T, thioflavin S and derivatives thereof.
  • the fluorescent probe contains a quantum dot as a fluorescent dye or is a quantum dot.
  • the maximum fluorescence wavelength of the fluorescent probe is in the range of 500 to 610 nm.
  • the concentration of the test substance is 0.1 mg/ml or less.
  • a system for determining amyloid formation inhibitory activity of a test substance which comprises an imaging device and a computer, Aggregation reaction formation obtained by reacting an amyloidogenic protein with a fluorescent probe capable of binding to amyloid formed by polymerization of the amyloidogenic protein in the presence or absence of a test substance
  • a device that captures the fluorescence of an object Computer
  • the fluorescence of the agglutination reaction product is calculated from the brightness value of each pixel included in the region of interest in the captured fluorescence image by the following equation 1:
  • Sum Intensity (%) (sum of brightness values of pixels in the region of interest / sum of theoretical maximum brightness values of pixels in the region of interest) x 100
  • a standard deviation calculation unit that calculates a standard deviation from the brightness value of each pixel included in the region of interest in the fluorescence image captured by the imaging device, The value of the standard deviation of
  • the activity determination unit determines that the test substance has an amyloid formation inhibitory activity when it is smaller than the value of the standard deviation of the brightness value of the above system.
  • the imaging device images the fluorescence of the agglutination reaction product in the presence of multiple concentrations of the test substance, the standard deviation calculation unit calculates the brightness value standard deviation corresponding to each test substance concentration, and the activity determination unit inhibits it.
  • the 50% effective concentration (EC 50 ) is further determined from an inhibition curve showing the relationship between the concentration of the test substance and the standard deviation of the luminance value, according to any one of (18) to (21). system.
  • amyloid formation of various proteins such as amyloid ⁇ , tau, and ⁇ -synuclein can be evaluated with high accuracy by reducing an experimental error depending on the skill of an experimenter and the type of sample. ..
  • a larger amount of samples can be evaluated in parallel with high precision in the trace high-throughput screening system, and amyloid formation inhibitory substances can be efficiently screened.
  • FIG. 3 shows fluorescence microscope images of amyloid ⁇ (A ⁇ ) aggregation reaction products in the presence of various concentrations of Lupinus luteus extract, and calculated EC 50 values of A ⁇ aggregation inhibitory activity. Fluorescence spectra of Qdot605 and Qdot655 are shown in the upper part, and absorption spectra of Rosa rugosa extract are shown in the lower part. Using fluorescence-labeled A ⁇ with Qdot 605 or Qdot 655 as a fluorescent probe, a fluorescence microscope image of the A ⁇ aggregation reaction product in the presence of various concentrations of rosmarinic acid and the calculated EC 50 value of the A ⁇ aggregation inhibitory activity were obtained. Show.
  • FIG. 11a shows a fluorescence microscope image of the A ⁇ aggregation reaction product in the presence of various concentrations of DMSO
  • FIG. 11b shows a relationship between the DMSO concentration and the brightness value SD calculated from the fluorescence microscope image of the A ⁇ aggregation reaction product. ..
  • SD is represented by a relative value when the value of the luminance value SD in the presence of 2.5% DMSO is 100%.
  • FIG. 12a shows an image showing the range of the region of interest occupying the well containing the A ⁇ aggregation reaction product
  • FIG. 12b shows the relationship between the size of the region of interest and the brightness value SD of the pixels contained in the region of interest.
  • Fig. 13a shows a fluorescence microscope image of the A ⁇ agglutination reaction product imaged at various exposure times, and a luminance value histogram of pixels included in the region of interest of each image
  • Fig. 13b shows Sum Intensity calculated from the fluorescence microscope image. The relationship with the brightness value SD is shown.
  • FIG. 15a shows a fluorescence microscope image of the A ⁇ aggregation reaction product imaged with various camera gains
  • FIG. 15b shows the relationship between the gain and the brightness value SD calculated from the fluorescence microscope image.
  • FIG. 3 is a diagram in which the well number of the 1536 well plate on which the A ⁇ aggregation reaction was performed and the brightness value SD calculated from the fluorescence microscope image of the well are plotted.
  • FIG. 6 is a plot of EC 50 values of A ⁇ aggregation inhibitory activity calculated by 61 sets of A ⁇ aggregation inhibitory activity evaluation tests using rosmarinic acid as a test substance. It is a figure which shows the structure activity relationship of a tropolone derivative.
  • MA is EC 50 values calculated by the A ⁇ aggregation inhibitory activity evaluation system of the present invention
  • TA is the a EC 50 values calculated by the conventional Thioflavin T (ThT) assay.
  • FIG. 19a shows a fluorescence microscope image (top) and a confocal laser scanning microscope image (bottom) of tau aggregation reaction products in the presence of various concentrations of dithiothreitol (DTT), and FIG. 19b shows DTT concentration and tau aggregation reaction production.
  • FIG. 19c shows the relationship between the brightness value SD calculated from the fluorescence microscope image of the product and the fluorescence microscope image (top) and the confocal laser microscope image (bottom) of the tau aggregation reaction products at various tau MDB fragment concentrations.
  • FIG. 19d shows the relationship between the tau MDB fragment concentration and the brightness value SD calculated from the fluorescence microscopic image of the tau aggregation reaction product, and FIG.
  • FIG. 20e shows the supernatant obtained by the tau sedimentation assay in the presence or absence of DTT.
  • the photograph of (S) and the pellet (P) of SDS-PAGE is shown.
  • Structures of tau (upper row) and tau MDB fragment (lower row) are shown in FIG. 20a
  • TEM images of tau aggregation reaction product and A ⁇ aggregation reaction product in the absence of fluorescent probe are shown in FIG. 20b
  • fluorescent probe is shown in FIG. 20c.
  • 20d shows a fluorescence microscope image of both agglutination products taken with time
  • FIG. 20d shows a TEM image of both agglutination products after the reaction in the presence of a fluorescent probe
  • FIG. 20d shows a fluorescence microscope image of both agglutination products after the reaction in the presence of a fluorescent probe
  • FIG. 20f A confocal laser microscope image of both agglutination products obtained with time is shown in Fig. 20f
  • a fluorescence microscope image of both agglutination products after the reaction in the presence of a fluorescent probe is shown in Fig. 20f
  • a fluorescent probe is shown in Fig. 20g.
  • Fig. 3 shows the relationship between the luminance value SD calculated from the fluorescence microscope images of both agglutination reaction products and the agglutination time.
  • FIG. 3 shows TEM images and fluorescence microscope images of A ⁇ aggregation reaction products, tau aggregation reaction products, and ⁇ -synuclein aggregation reaction products in the absence and presence of fluorescent probes when quantum dots were used as fluorescent probes.
  • FIG. 24A shows a fluorescence microscope image of the A ⁇ aggregation reaction product when ThT was used as a fluorescence probe
  • FIG. 24B shows the brightness value SD calculated from the fluorescence microscope image.
  • the first aspect of the present invention in the presence or absence of a test substance, amyloidogenic protein, and the binding ability to amyloid formed by polymerization of the amyloidogenic protein, An agglutination reaction step of reacting with a fluorescent probe that has; an imaging step of imaging the fluorescence of the agglutination reaction product obtained in the agglutination reaction step, the brightness value of each pixel included in the region of interest in the imaged fluorescence image
  • Sum Intensity (%) (sum of brightness values of pixels in the region of interest / sum of theoretical maximum brightness values of pixels in the region of interest) x 100
  • the test substance comprises an activity determination step of determining that it has amyloid formation inhibitory activity, and the amyloid formation inhibitory activity of the test substance is determined. Regarding the method of determination.
  • FIG. 1 shows each step in the method of one embodiment
  • FIG. 2 shows details of the activity determination step
  • FIG. 3 shows each step in the method of another embodiment for performing luminance value correction
  • FIG. 5 shows steps in a method of another embodiment for performing luminance value correction in some cases
  • FIG. 5 shows steps in a method of yet another embodiment for calculating an EC 50 of a test substance.
  • the agglutination step has the ability to bind to amyloid-forming protein and amyloid formed by polymerization of the amyloid-forming protein in the presence or absence of a test substance. This is a step of reacting with a fluorescent probe.
  • amyloid is formed by polymerization of the amyloidogenic protein, and the amyloid and the fluorescent probe are bound simultaneously with the amyloid formation or after the amyloid formation.
  • amyloid formation inhibition and “amyloid aggregation inhibition” are used interchangeably in this specification.
  • amyloidogenic protein used in the present invention is a protein having the ability to form amyloid by polymerization, and is preferably an amyloidogenic protein of biological origin that causes amyloidosis, a partial peptide thereof or a derivative thereof. ..
  • Amyloidogenic proteins are known to those skilled in the art, examples of which are amyloid ⁇ protein, tau, ⁇ -synuclein, prion protein, Huntington, amylin, apolipoprotein AI, apolipoprotein A-II, serum amyloid A, immunoglobulin.
  • Light chain immunoglobulin heavy chain, ⁇ 2-microglobulin, insulin, lysozyme, cystatin C, stefin A, transthyretin, gelsolin, atrial natriuretic factor, keratoepithelin, lactoferrin and the like can be mentioned.
  • the amyloidogenic protein may be selected according to the target amyloidosis.
  • Table 1 shows typical examples of amyloidosis and the amyloids that are the causes thereof.
  • a partial peptide of an amyloidogenic protein is a fragment of the amyloidogenic protein that retains the amyloidogenic ability of the original amyloidogenic protein.
  • a derivative of an amyloidogenic protein or a partial peptide thereof means a modified form of the amyloidogenic protein or a partial peptide thereof, which retains the amyloidogenic ability of the original amyloidogenic protein, and is typically amyloidogenic. It is a variant of the sex protein or its partial peptide. Preferred examples of the variant include one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and most preferably 1 in the amino acid sequence of the amyloidogenic protein or its partial peptide.
  • a protein or peptide consisting of an amino acid sequence in which two amino acid residues are deleted or substituted, or one or more, for example 1 to 10, preferably 1 to 1 amino acid sequence in the amino acid sequence of an amyloidogenic protein or its partial peptide
  • a protein or peptide having an amino acid sequence to which 5 amino acid residues, more preferably 1 to 3 amino acid residues, and most preferably 1 or 2 amino acid residues are added, and having amyloid-forming ability is exemplified. it can.
  • the substitution is preferably a so-called conservative substitution, and examples thereof include glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid ( Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), threonine and serine (Ser) or alanine, lysine (Lys) and arginine (Arg), and other amino acids
  • conservative substitution examples thereof include glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid ( Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine (
  • the identity is calculated using the amino acid sequence of the amyloidogenic protein or its partial peptide
  • the initial condition parameters of BLAST at least 70% or more, preferably 80% or more, more preferably 90% or more, More preferably 95% or more, particularly preferably 97%, 98% or 99% or more protein or peptide consisting of an amino acid sequence having an identity, and the protein having amyloidogenic ability is also an amyloidogenic protein. Or it is included in the variant of the partial peptide.
  • the amyloidogenic protein is not limited in its length as long as it has the ability to form amyloid, and is disclosed in, for example, JP-A-2017-007990 (which is incorporated herein by reference in its entirety). It may be an oligopeptide consisting of 10 amino acids such as N-terminal peptide and C-terminal peptide of protein A-II, or may be a polypeptide consisting of tens amino acids such as amyloid ⁇ protein. , A polypeptide consisting of hundreds of tens of amino acids, such as a fragment containing four repeating sequences of the tau microtubule binding domain (MDB), or consisting of hundreds of amino acids such as the full length of tau It may be a polypeptide.
  • the term peptide includes a polypeptide, and a protein and a peptide are used interchangeably.
  • amyloidogenic protein preferably used in the present invention is preferably selected from the group consisting of amyloid ⁇ protein, tau and ⁇ -synuclein and partial peptides thereof and derivatives thereof, and particularly preferably amyloid ⁇ 42 (SEQ ID NO: 1), a fragment (SEQ ID NO:3) or a ⁇ -synuclein (SEQ ID NO:4) containing a four-repeat sequence of Tau MDB.
  • the fluorescent probe used in the present invention is a fluorescent substance capable of binding to amyloid formed by polymerization of amyloid-forming protein.
  • the ability to bind to amyloid means the ability to form amyloid together with the amyloidogenic protein, or the ability to bind to amyloid formed by polymerization of the amyloidogenic protein.
  • the fluorescent probe used in the present invention is a fluorescent substance capable of binding to amyloid in a form incorporated in amyloid by forming amyloid together with an amyloidogenic protein (hereinafter, also referred to as “amyloidogenic fluorescent substance”). ), or a fluorescent substance capable of binding to amyloid formed by polymerization of amyloid-forming protein (hereinafter, also referred to as “amyloid-binding fluorescent substance”).
  • An amyloid-forming fluorescent substance is a substance in which a substance capable of forming amyloid together with an amyloid-forming protein, typically a peptide capable of forming amyloid together with an amyloid-forming protein, is fluorescently labeled.
  • This peptide is not limited in its sequence or length as long as it has the ability to form amyloid together with the amyloidogenic protein, and may be the same as or different from the amyloidogenic protein.
  • This peptide is preferably an amyloidogenic protein of biological origin, a partial peptide thereof or a derivative thereof. Further, it may be an artificially designed peptide as disclosed in Table 1 of JP-A-2017-007990.
  • the combination of the amyloidogenic protein preferably used in the present invention and the peptide having the ability to form amyloid together with the amyloidogenic protein is a combination of amyloid ⁇ 42 (SEQ ID NO: 1) and amyloid ⁇ 40 (SEQ ID NO: 2), Alternatively, it is a combination of fragments (SEQ ID NO: 3) each containing a tau MDB four-repeat sequence.
  • the amyloid-binding fluorescent substance may be any fluorescent substance that does not affect the polymerization and aggregation of amyloid-forming protein and can bind to the formed amyloid, typically, an amyloid-binding fluorescent dye, or It is a fluorescently labeled amyloid-binding substance.
  • the amyloid-binding fluorescent dye is a fluorescent dye capable of specifically or nonspecifically binding to amyloid, and is preferably a fluorescent dye having an affinity for a ⁇ -sheet structure.
  • amyloid-binding fluorescent dyes known to those skilled in the art include Congo red, thioflavin T, thioflavin S and their derivatives such as (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl). -4-hydroxy)styrylbenzene (BSB), (trans,trans )-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), publicly known papers (eg Masato Higuchi, Dozin News No. 115, pages 1 to 9, published on July 11, 2005, Dojindo Laboratories Co., Ltd.). Quantum dots can also be used as an amyloid-binding fluorescent dye.
  • Quantum dots are nano materials with a three-dimensional quantum confinement structure, and semiconductor quantum dots, carbon-based quantum dots, etc. are known. Due to their excellent fluorescence performance, quantum dots are being used in a wide range of fields including bioimaging as an alternative to conventional fluorescent dyes.
  • Examples of quantum dots that are preferably used as amyloid-binding fluorescent dyes in the present invention include semiconductor quantum dots, especially core-shell CdSe/ZnS quantum dots Qdot (registered trademark) 525, Qdot545, Qdot565, Qdot585, Qdot605. , Qdot655, Qdot705 and Qdot800 (all Thermo Fisher Scientific) and the like, and Qdot605 or Qdot655, particularly Qdot605 is preferable.
  • Quantum dots can be used as they are or in the state of being polymer coated.
  • the size and shape of the quantum dots are not limited, but the diameter thereof is preferably 1 nm to 100 nm, preferably 1.5 nm to 50 nm, more preferably 2 nm to 20 nm.
  • the quantum dot increases, the fluorescent wavelength shifts to the long wavelength side. Therefore, when the quantum dot is used as the fluorescent dye, the quantum dot having the particle size corresponding to the desired fluorescent wavelength is selected.
  • the fluorescently labeled amyloid-binding substance is typically a fluorescently labeled anti-amyloid-specific antibody.
  • the anti-amyloid-specific antibody that is, an antibody that specifically binds to amyloid means an antibody that preferentially binds to a target amyloid over a non-target protein, and that the antibody does not bind to a non-target protein at all. It does not mean.
  • An anti-amyloid-specific antibody can also be described as an antibody that has a high binding affinity for the target amyloid and is at least 2-fold stronger than its affinity with the non-target protein, preferably at least 10-fold stronger, more preferably at least 20-fold stronger. , Most preferably at least 100-fold stronger affinity, capable of binding to the target amyloid.
  • the antibody may be a monoclonal antibody or a polyclonal antibody, and Fab, Fab', F(ab')2, scFv or F(ab')2 antibody fragments derived from these antibodies, diabodies. It may be a peptide containing (diabody), dsFv, CDR or the like.
  • the fluorescent dye used for the fluorescent labeling of each of the above substances may be a fluorescent dye that does not affect the polymerization and aggregation of the amyloidogenic protein, for example, a general organic fluorescent dye such as fluorescein and rhodamine. Good, but inorganic fluorescent dyes, especially quantum dots, are preferred. Examples of suitable quantum dots are as described above.
  • the fluorescent label may be a known method depending on the fluorescent dye used, for example, a sulfhydryl-reactive crosslinking agent (thiol-maleimide crosslinking), a carboxyl-amine crosslinking agent (primary amine-carbodiimide crosslinking), an amine-reactive crosslinking agent. (Crosslinking of primary amine-NHS ester or imide ester) and the like.
  • a sulfhydryl-reactive crosslinking agent thiol-maleimide crosslinking
  • a carboxyl-amine crosslinking agent primary amine-carbodiimide crosslinking
  • an amine-reactive crosslinking agent Rosslinking of primary amine-NHS ester or imide ester
  • the fluorescent probe retains the ability to bind to amyloid and is capable of fluorescence detection, there is no limitation on the ratio of the amount of the fluorescent dye and the labeled substance in the fluorescent label, and there are multiple molecules of the labeled substance
  • the fluorescent wavelength emitted by the fluorescent probe is not limited, when using a test substance derived from a plant, the maximum fluorescent wavelength of the fluorescent probe is preferably in the range of 500 ⁇ 660 nm or 700 ⁇ 800 nm, in particular, It is preferably in the range of 500 to 610 nm. Further, the excitation wavelength for exciting the fluorescent probe is appropriately determined according to the characteristics of the fluorescent dye used.
  • the agglutination reaction is performed under reaction conditions that allow the amyloid-forming protein to polymerize to form an aggregate.
  • the concentration of the amyloidogenic protein at the start of the agglutination reaction is about 1 to 100 ⁇ M, preferably 10 to 50 ⁇ M, as the final concentration in the reaction solution.
  • the concentration of the fluorescent probe is about 0.005-0.5%, preferably 0.01-0.1% of the amyloidogenic protein concentration.
  • concentrations of the amyloidogenic protein and the fluorescent probe can be appropriately set depending on the types and properties of the amyloidogenic protein and the fluorescent probe used and the imaging conditions such as the objective lens magnification.
  • amyloidogenic fluorescent substance as a fluorescent probe
  • the amyloidogenic protein is amyloid ⁇ 42 and the peptide capable of forming amyloid together with the amyloidogenic protein is amyloid ⁇ 40
  • the concentration of the fluorescent probe is preferably 25 ⁇ M and 25 nM, respectively.
  • the concentrations of the amyloidogenic protein and the fluorescent probe are 10 ⁇ M and It is preferably 50 nM.
  • the agglutination reaction is carried out in the presence or absence of the test substance in an appropriate buffer solution such as water or PBS.
  • the buffer may contain 10% or less of DMSO and 20% or less of EtOH.
  • concentration is preferably 0.1 mg/ml or less, particularly preferably 0.000001 to 0.1 mg/ml.
  • Precipitation may occur if a crudely purified substance such as a plant extract is used in a high concentration in the agglutination reaction.Therefore, for test substance concentrations of 0.1 mg/ml or less, it is possible to omit extra precipitation removal operations such as centrifugation. It is useful because it can be done.
  • the agglutination reaction can be performed in the well of a microplate generally used for fluorescence observation.
  • the liquid level of the agglutination reaction liquid and the bottom of the well where the agglutinates are deposited are The bottom shape of is preferably flat.
  • a flat-bottom microplate of 1536 wells or the like that can evaluate more test substances at once Is preferably used.
  • the temperature of the agglutination reaction is about room temperature to 37°C, preferably 37°C, and the reaction time is about 4 to 36 hours, preferably 12 to 24 hours. Shaking or stirring may excessively promote the formation of aggregates and may adversely affect the determination of the amyloid formation inhibitory activity. Therefore, the aggregation reaction is preferably performed in a stationary state.
  • the fluorescence of the agglutination reaction product obtained in the agglutination reaction step is calculated from the brightness value of each pixel included in the region of interest in the captured fluorescence image Sum. This is a step of imaging under an exposure condition where the intensity is 15 to 85%.
  • the fluorescence of the agglutination reaction product can be imaged using an imaging device connected to a computer that controls operations such as setting and control of imaging conditions and imaging and displaying of imaging data.
  • the imaging device is typically an epi-illumination fluorescence microscope equipped with a CCD camera, in which a container accommodating the agglutination reaction product, for example, a microwell plate, is set and irradiated with excitation light to cause fluorescence from the agglutination reaction product. Illuminate and take an image with a CCD camera.
  • the excitation light can be appropriately determined according to the characteristics of the fluorescent dye used.
  • the wavelength of the excitation light may be shorter than 580 nm, for example 532 to 552 nm.
  • the bandpass filter for selectively transmitting the generated fluorescence to capture an image of the fluorescence may be selected so that only the light in the wavelength band including the fluorescence wavelength of the fluorescent dye used can be selectively transmitted,
  • a bandpass filter capable of selectively transmitting only light in the wavelength band of 594 to 646 nm can be used as an example.
  • the fluorescence of the agglutination reaction product is imaged by the following Expression 1: from the luminance value of each pixel included in the region of interest in the captured fluorescence image.
  • Sum Intensity (%) (sum of brightness values of pixels in the region of interest / sum of theoretical maximum brightness values of pixels in the region of interest) x 100 It is performed by controlling the exposure so that the Sum Intensity calculated by is 15 to 85%.
  • Imaging under exposure control based on Sum Intensity is specifically performed as follows. First, an arbitrary region of interest is set in the fluorescence image of the agglutination reaction product preliminarily imaged under an appropriate exposure condition, and the brightness value information of each pixel contained therein is acquired.
  • the fluorescence image may be color or monochrome. In the case of a color image, it is possible to obtain luminance value information by performing grayscale conversion from the RGB value of each pixel by the following formula.
  • Luminance value Y 0.299 ⁇ R+0.587 ⁇ G+0.114 ⁇ B
  • Sum Intensity (sum of luminance values of all 186,624 pixels included in the region of interest / (186,624 x 255)) x 100
  • the number of bits of the fluorescence image is preferably 8 bits. Although it is possible to use an image with a bit number larger than 8 bits, it is preferable to calculate the Sum Intensity after converting to an 8-bit image because the image processing load is heavy.
  • Imaging is performed by referring to the Sum Intensity from the preliminary fluorescence image calculated in this way and controlling the exposure so that the Sum Intensity becomes a predetermined set value.
  • the exposure can be controlled by adjusting the exposure time and camera gain (ISO sensitivity) and using a neutral density filter, in particular by adjusting the exposure time and camera gain (ISO sensitivity) appropriately. It is preferable to utilize the function. For example, when the Sum Intensity calculated from the preliminary fluorescence image is below a predetermined set value, the Sum Intensity can be increased by lengthening the exposure time and/or increasing the camera gain.
  • the Sum Intensity calculated from the preliminary fluorescence image exceeds the predetermined set value
  • the Sum Intensity can be reduced by shortening the exposure time and/or reducing the camera gain.
  • the setting value of SumIntensity can take any value within the range of 15 to 85%, and is preferably set within the range of 45 to 65%.
  • the above preliminary fluorescence image can be captured immediately before capturing the fluorescence of the agglutination reaction product used for determination, so that the value of SumIntensity is referenced so that SumIntensity becomes a predetermined set value.
  • the exposure can be feedback controlled.
  • the preliminary fluorescence image may be captured before the determination method is started, and the determination method may be started after determining the exposure condition at which the SumIntensity becomes a predetermined set value.
  • the exposure condition that the Sum Intensity is 15 to 85% is 150 ms to 1.8 s, preferably 300 ms to 1.6 at ISO sensitivity 200 equivalent (1x gain for a camera equivalent to ISO sensitivity 200).
  • Exposure conditions for exposure is 150 ms to 1.8 s, preferably 300 ms to 1.6 at ISO sensitivity 200 equivalent (1x gain for a camera equivalent to ISO sensitivity 200).
  • the exposure condition at which the Sum Intensity is 45 to 65% is the exposure condition of 500 to 900 ms of exposure at an ISO sensitivity of 200.
  • use the automatic exposure function of the CCD camera to set the target maximum brightness to 50% and perform exposure at an ISO sensitivity equivalent to 200 for a maximum exposure time of 160 ms, and then set the target maximum brightness to 50% within that exposure time.
  • the exposure condition in which the ISO sensitivity is gradually increased to 6400 equivalent is also preferably used.
  • the target maximum brightness is the percentage of the brightness value of the brightest pixel among the pixels included in the imaging range with respect to the camera gradation. For example, when a target maximum brightness is set to 50% in a 256-gradation camera capable of capturing an 8-bit image, imaging is performed so that the brightness value of the brightest pixel in the pixels included in the imaging range becomes 128. .. At this time, the allowable amount of over-illumination may be set appropriately.
  • the SD calculation step is a step of calculating SD from the brightness value of each pixel included in the region of interest in the fluorescence image captured in the imaging step.
  • the fluorescence image used here may be color or monochrome, and in the case of a color image, the RGB value of each pixel is grayscale converted, and it must be an 8-bit image. Is preferred.
  • This correction may be performed on all fluorescence images (steps S22 and S23), but from the viewpoint of more accurately evaluating the amyloid formation inhibitory activity by increasing the value of the brightness value SD, the above formula 1 is used.
  • the SD is calculated after correcting the fluorescence image whose Sum Intensity calculated by is smaller than the target Sum Intensity, and the SD is calculated, and the fluorescence image whose Sum Intensity is greater than the target Sum Intensity is not corrected. It is preferable to calculate SD using the brightness value as it is (steps S32 to S35).
  • the activity determination step compares the value of the brightness value SD in the presence of the test substance calculated in the SD calculation step with the value of the brightness value SD in the absence of the test substance, This is a step of determining that the test substance has amyloid formation inhibitory activity when the value of the brightness value SD in the presence of the test substance is smaller than the value of the brightness value SD in the absence of the test substance.
  • the present invention by utilizing the fact that the brightness value SD calculated from the fluorescence image of the amyloid aggregates is positively correlated with the amount of amyloid aggregation, evaluation of amyloid formation is performed using the value of the brightness value SD as an index. .. Therefore, when the value of the brightness value SD in the presence of the test substance is smaller than the value of the brightness value SD in the absence of the test substance, the amount of amyloid aggregation is small in the presence of the test substance, that is, the test substance does not form amyloid. It can be determined that it has inhibitory activity (steps S131 and S132).
  • the present invention also includes the agglutination reaction step, the imaging step and the SD calculation step described above, and the brightness value SD in the presence of the test substance calculated in the SD calculation step and the brightness value SD in the absence of the test substance.
  • the activity determination step to determine that the test substance has amyloid formation promoting activity is performed.
  • a method for determining the activity of promoting amyloid formation of a test substance is provided.
  • the amyloid aggregation amount does not change even in the presence of the test substance, that is, It can be determined that the test substance has neither amyloid formation inhibitory activity nor amyloid formation promoting activity (steps S135 and S136).
  • agglutination reaction is performed using multiple concentrations of the test substance in the agglutination reaction step, and the brightness value SD corresponding to each test substance concentration is calculated in the SD calculation step, and in addition to the inhibitory activity determination in the activity determination step. It is also possible to create an inhibition curve showing the relationship between the concentration of the test substance and the luminance value SD, and use this to determine the 50% effective concentration (EC 50 ) (steps S44 and S45). Also included in the present invention.
  • the present invention also comprises an agglutination step of reacting a sample with a fluorescent probe capable of binding to amyloid formed by polymerization of an amyloidogenic protein; a mixture of the sample and the fluorescent probe before and after the reaction in the agglutination step.
  • the imaging step for imaging the fluorescence of the mixture wherein the mixture is exposed under the exposure condition that the Sum Intensity calculated from the brightness value of each pixel included in the region of interest in the captured fluorescence image is 15 to 85%.
  • the method for evaluating amyloid formation in a sample includes the step of determining amyloid formation in which it is determined that amyloid is formed when the value of is larger than the value of the brightness value SD before the agglutination reaction.
  • Another aspect of the present invention is a system comprising an imaging device and a computer for determining amyloid formation inhibitory activity of a test substance, Aggregation reaction formation obtained by reacting an amyloidogenic protein with a fluorescent probe capable of binding to amyloid formed by polymerization of the amyloidogenic protein in the presence or absence of a test substance
  • a device that captures the fluorescence of an object The computer images the fluorescence of the agglutination reaction product under the exposure condition that the Sum Intensity calculated from the brightness value of each pixel included in the region of interest in the captured fluorescence image is 15 to 85% calculated by the above formula 1.
  • Control unit for controlling the image pickup device; an SD calculation unit for calculating SD from the luminance value of each pixel included in the region of interest in the fluorescence image captured by the image pickup device; and the luminance value SD in the presence of the test substance Value is compared with the value of the brightness value SD in the absence of the test substance, and the value of the brightness value SD in the presence of the test substance is less than the value of the brightness value SD in the absence of the test substance
  • Relates to the above-mentioned system which comprises an activity determination unit for determining that it has amyloid formation inhibitory activity.
  • the "system for determining the amyloid formation inhibiting activity of a test substance” is used interchangeably with the "device for determining the amyloid formation inhibiting activity of a test substance”.
  • the system 1 is composed of an imaging device 10 and a computer 20.
  • the image pickup device 10 is typically a general epi-fluorescence microscope device having an objective lens, an eyepiece lens, an imaging lens, a light source, an excitation filter, a dichroic mirror, an absorption filter and a CCD camera as a basic configuration.
  • the CCD camera captures a fluorescence image under the exposure condition that the control unit 26 controls the Sum Intensity to be 15 to 85%.
  • the fluorescence image is sent to the image input unit 22 immediately after being captured or after being temporarily recorded in a storage device such as a server.
  • the computer 20 has an input unit 21 for inputting instructions and information to the computer 20, an image input unit 22 for inputting fluorescence image data from the image pickup device 10, a recording unit 23, and a predetermined unit for the captured fluorescence image data.
  • An arithmetic unit 24 that performs arithmetic processing, an output unit 25 that outputs a fluorescence image and an arithmetic result, and a control unit 26 that controls the operation of each of the above units.
  • the system 1 is further connected to a sample mixing device for mixing components such as a fluorescent probe used in the agglutination reaction, an agglutination reaction device for reacting the mixed sample, and a display unit for displaying information output from the output unit 25. You can also
  • the input unit 21 is a device such as a keyboard, a mouse, a button, and a switch, and inputs signals to the control unit 26 in accordance with operations on these.
  • the image input unit 22 is an interface for inputting image data from a camera attached to the imaging device 10 or a storage device such as a server that stores fluorescence image data.
  • the recording unit 23 includes a recording medium such as a hard disk, a flash memory, a RAM, and a ROM, and a reading device that reads information recorded on the recording medium in its configuration.
  • the recording unit 23 is image data input from the image input unit 22, a program for image processing and activity determination executed by the arithmetic unit 24, a program for controlling the operations of the above units and devices executed by the control unit 26. , Records various setting information and the like.
  • the program is recorded in the program recording unit 231.
  • Calculator 24 is configured by hardware such as CPU.
  • the calculation unit 24 reads the program recorded in the program recording unit 231, and executes image processing on the fluorescence image data recorded in the recording unit 23.
  • the activity determining unit 242 for determining the amyloid formation inhibitory activity is included in the configuration.
  • the calculation unit 24 may include a SumIntensity calculation unit that calculates the SumIntensity from a fluorescence image that is preliminarily captured for controlling the exposure condition.
  • the control unit 26 controls the exposure of the imaging device 10 based on the value of the SumIntensity calculated by the SumIntensity calculation unit so that the SumIntensity calculated from the captured fluorescence image is 15 to 85%.
  • the calculation unit 24 further determines the brightness value correction unit that corrects the brightness value from the fluorescent image captured under the controlled exposure condition to obtain the corrected brightness value, and whether to perform the correction in the brightness value correction unit. It may include a SumIntensity determining unit that determines whether the SumIntensity calculated from the brightness value information for determining is a value equal to or higher than the target SumIntensity.
  • the activity determination unit 242 can also determine the amyloid formation promoting activity of the test substance and evaluate the amyloid formation in the sample.
  • the output unit 25 outputs information such as a fluorescence image captured by the imaging device 10, various numerical values calculated by the arithmetic unit 24, and activity determination results to the outside of the system.
  • the output unit 25 may be connected to a display unit such as a display or an external storage device.
  • the control unit 26 is connected to the above-described units and devices, reads the program recorded in the program recording unit 231, and controls these operations.
  • the control unit 26 is also connected to and can control the sample mixing device and the agglutination reaction device.
  • the system of this aspect can be used to implement the determination method of the first aspect, and the details are as described for the above determination method.
  • a further aspect of the present invention is a program operating on a computer for determining amyloid formation inhibitory activity of a test substance, the computer comprising: Fluorescence of the agglutination reaction product obtained by reacting the amyloidogenic protein in the presence or absence of the test substance and a fluorescent probe having the ability to bind to amyloid formed by the polymerization of the amyloidogenic protein
  • a control unit for controlling the image pickup device so as to pick up an image under an exposure condition in which the Sum Intensity calculated by the equation 1 from the luminance value of each pixel included in the region of interest in the imaged fluorescence image is 15 to 85%.
  • An SD calculation unit that calculates SD from the brightness value of each pixel included in the region of interest in the fluorescence image captured by the imaging device, The brightness value SD in the presence of the test substance is compared with the brightness value SD in the absence of the test substance, and the brightness value SD in the presence of the test substance is the brightness value in the absence of the test substance.
  • the present invention relates to a program for causing a test substance to function as an activity determination unit that determines that it has an amyloid formation inhibitory activity when it is smaller than SD.
  • the program may be a program for causing the computer to further function as a Sum Intensity calculation unit that calculates Sum Intensity from a fluorescence image preliminarily captured for controlling the exposure condition.
  • the program corrects the brightness value from the fluorescence image taken under the exposure condition determined to have Sum Intensity of 15 to 85% to obtain a corrected brightness value by the program.
  • SumIntensity judgment that determines whether the SumIntensity calculated from the brightness value information to determine whether to perform correction in the brightness value correction unit and/or in the brightness value correction unit is greater than or equal to the target SumIntensity It may be a program for further functioning as a unit.
  • the program can cause the computer to function as an activity determination unit that determines amyloid formation promoting activity of a test substance and evaluates amyloid formation in a sample.
  • the program can cause the computer to function as a control unit that further controls the mixing of components such as the fluorescent probe used in the agglutination reaction and the reaction of the mixed sample.
  • the program of this aspect can be used to operate a computer as the system of the above aspect, and details thereof are as described in the above determination method and system.
  • the present invention also provides, as another aspect, a recording medium that records the above program in a computer-readable manner.
  • the recording medium may be a portable medium such as a CD (Compact Disc), a DVD (Digital Versatile Disc), a USB flash drive, or a storage device such as a hard disk built in the computer.
  • the program recorded on the recording medium may be for realizing all or some of the functions described above, and in the latter case, the program is already recorded on the computer.
  • the functions described above may be realized by a combination with an existing program.
  • a substance having amyloid formation inhibiting activity can be obtained.
  • the substance having an amyloid formation inhibitory activity can be used as an active ingredient of a medicine and food and drink for the prevention and/or treatment of amyloidosis, and the present invention also provides such a medicine and food and drink.
  • the medicine and food and drink can be a composition containing other components.
  • the food and drink are beverages (including concentrated stock solution of beverage and powder for adjustment), general processed foods containing seasonings, supplements, health foods (functional foods and drinks), foods with health claims (foods for specified health use, It is understood to include a wide range of nutritionally functional foods and foods with functional claims.
  • the pharmaceutical and food and drink provided by the present invention are subjects suffering from or at risk of suffering from amyloidosis, such as mice, rats, hamsters, rodents including guinea pigs, humans, chimpanzees, primates including rhesus monkeys, pigs, It is administered to mammals such as cattle, goats, horses, domestic animals including sheep, dogs, and pets including cats.
  • the preferred subject is a human.
  • the medicine and food and drink provided by the present invention contain an effective amount of an amyloid formation inhibitor.
  • the effective amount is an amount that can inhibit amyloid formation in the administered subject, and is appropriately adjusted depending on the strength of inhibitory activity, administration route, type of disease, severity of symptoms, patient and other medical factors. To be done.
  • the effective amount can be administered once or several times a day, or can be administered or ingested intermittently.
  • the medicine provided by the present invention can contain a pharmaceutically acceptable carrier or additive, or a physiologically active substance other than the active ingredient.
  • Pharmaceutically acceptable carriers or additives are well known to those skilled in the art, and those skilled in the art can use the ingredients described in the 17th Revised Japanese Pharmacopoeia and other standards within a range of ordinary practicing ability. It can be appropriately selected and used according to the form.
  • the medicine provided by the present invention can take any dosage form.
  • dosage forms include tablets, capsules, granules, fine granules, oral liquids, powders, syrups and other oral dosage forms, suppositories, ointments, creams, gels, patches, sprays and lotions.
  • the medicament provided by the present invention can be administered by any administration route known to those skilled in the art, for example, parenteral administration such as oral administration, topical administration or intravenous administration. Manufacture of such a drug may be carried out by using a general method known or well known to those skilled in the art.
  • the medicine, food and drink provided by the present invention can be used for the prevention and/or treatment of amyloidosis. Therefore, the present invention also includes a method of preventing and/or treating amyloidosis, which comprises administering to a subject in need thereof an effective amount of an effective amount of an amyloidogenesis inhibitor.
  • Example 1 Establishment of automated amyloid ⁇ aggregation inhibitory activity evaluation system (1) Material and method Preparation of fluorescent probe QDA ⁇ Fluorescent probe QDA ⁇ labeled with quantum dots of human amyloid ⁇ (A ⁇ ) protein has been reported previously (Tokuraku K. et. al., PLoS ONE, 4, e8492, 2009 and Ishigaki Y. et. al. , PLoS ONE, 8, e72992, 2013, which are hereby incorporated by reference in their entirety), according to QD-PEG-NH 2 (Qdot® 655 ITK® Amino (PEG).
  • Quantum Dots Q21521MP or Qdot (registered trademark) 605 ITK (trademark) Amino (PEG) Quantum Dots; Q21501MP, both of which were prepared using Thermo Fisher Scientific). Briefly, 10 ⁇ M QD-PEG-NH 2 was first reacted with 1 mM sulfo-EMCS (22307, Pierce) in PBS for 1 h at room temperature. After unreacted sulfo-EMCS was removed by quenching, sulfo-EMCS bound to QD-PEG-NH 2 was added to 100 ⁇ M Cys-conjugated human A ⁇ 40 (23519, Anaspec: SEQ ID NO: 2) containing 5 mM EDTA.
  • QDA ⁇ (QD655A ⁇ , QD605A ⁇ ) was prepared by reacting with the PBS solution of 1) at room temperature for 1 hour. The concentration of QDA ⁇ was determined by comparing the absorbance at 350 nm with unlabeled QD-PEG-NH 2 .
  • test sample The dilution of the test sample and the dispensing of each solution used for the A ⁇ agglutination reaction were performed using an automated liquid dispensing robot system Automated Workstation JANUS G3 (Perkin Elmer).
  • Dilution Buffer (10% EtOH, 1xPBS) was aspirated using a conductive tip capable of detecting liquid levels (aspiration rate: 20 ⁇ l/sec) and a 384-well plate ( 384 Hard-Shell microplate HSP3951, BIO-RAD) was dispensed into each well (discharging height: 0.75 mm, discharging speed: 10 ⁇ l/sec).
  • Stock sample solutions were manually injected into the empty wells of a 384 well plate.
  • Aspirate the stock sample solution or the control solution (aspiration height: 2 mm, aspiration rate: 10 ⁇ l/sec), inject it into the adjacent well containing the dilution buffer (discharging height: 4 mm, ejection rate: 10 ⁇ l/sec), and mix by pipetting. (Suction height: 2 mm, Discharge height: 4 mm, Mixing speed: 25 ⁇ l/sec, Mixing frequency: 5).
  • This dilution step was repeated 5 times to prepare a 6-step dilution series for each sample.
  • ⁇ A ⁇ agglutination reaction Prepare 50 ⁇ M or 60 nM QDA ⁇ and 0-100 ⁇ M human A ⁇ 42 (4349-v, Peptide Institute: SEQ ID NO: 1) in PBS containing A ⁇ (containing 5% DMSO) at 4° C. Then, it was aspirated using a conductive tip capable of detecting the liquid level (aspiration rate: 30 ⁇ l/sec) and poured into a cooled 384-well plate at 4° C. (ejection height: 0.5 mm, ejection rate: 20 ⁇ l/sec).
  • the 1536 well plate was centrifuged at 1,530 ⁇ g for 5 minutes by a multi-plate centrifuge (PlateSpinII, Kubota) to remove air bubbles and level the liquid surface.
  • the 1536 well plate after centrifugation was incubated at 37° C. for 1 day to carry out A ⁇ agglutination reaction.
  • the final concentrations of QDA ⁇ and A ⁇ 42 in the agglutination reaction solution are 25 nM or 30 nM and 0 to 50 ⁇ M, respectively.
  • the concentration is expressed as the final concentration.
  • a typical plant crude extract, Lupinus luteus extract (concentration 0.0001-1 mg/ml) was incubated with 30 nM QD655 A ⁇ and 30 ⁇ M A ⁇ 42 to perform A ⁇ agglutination reaction, and automatic exposure function of CCD camera Fluorescence microscope images were taken using (AE) or under exposure conditions of 200 ms exposure time and 1x camera gain (Fig. 7).
  • the image taken under the exposure condition of exposure time 200ms and camera gain 1x showed the decrease of the overall brightness as the concentration of the test sample increased, and the brightness suitable for evaluation was not obtained especially at the sample concentration of 1 mg/ml. ..
  • FIG. 10 shows the relationship between the A ⁇ concentration and the brightness value SD.
  • the brightness value SD became maximum when the concentration of A ⁇ 42 was 25 ⁇ M.
  • the brightness value SD correlates with the amount of A ⁇ aggregates when the aggregate thickness is within the depth of focus (Ishigaki Y. et. al., supra). Used an A ⁇ 42 concentration of 25 ⁇ M unless otherwise noted.
  • a ⁇ agglutination reaction was performed by incubating 25 nM QD605A ⁇ and 25 ⁇ M A ⁇ 42 in the presence of solvent 2.5-10% DMSO, and fluorescence microscopy images were taken using the automatic exposure function of the CCD camera.
  • DMSO is used for preparing a stock solution of A ⁇ , and 2.5% corresponds to the DMSO concentration brought into the A ⁇ agglutination reaction solution.
  • the captured image is shown in FIG. 11a, and the relationship between the DMSO concentration and the brightness value SD of the pixels included in the region of interest of each image is shown in FIG. 11b. It was confirmed that there was no great difference in the brightness value SD at any DMSO concentration, and that DMSO up to a concentration of 10% did not affect the evaluation.
  • Region of interest Incubate 25 nM QD605A ⁇ and 25 ⁇ M A ⁇ 42 to perform A ⁇ agglutination reaction, and take fluorescence microscope image using CCD camera's automatic exposure function, 54 ⁇ 54 pixels in center of well, 216 ⁇ 216 pixels, 432 ⁇ 432 pixels, and 648 ⁇ 648 pixels were set in the ROI, and the brightness value SD of the pixels included in the ROI was calculated.
  • An image showing the range of the region of interest occupying the well is shown in FIG. 12a, and the relationship between the size of the region of interest and the brightness value SD is shown in FIG. 12b. It was confirmed that there was no big difference in the brightness value SD in any size of the ROI, and that the ROI of 54 ⁇ 54 pixels to 648 ⁇ 648 pixels could be set when the 1536 well plate was used.
  • the brightness value histogram has a normal distribution (white part in Fig. 13b), and the brightness value SD reached the peak during this exposure time.
  • Data from images taken using the automatic exposure feature of the CCD camera were also plotted within this range (gray circle in Figure 13b; AE).
  • the brightness value SD calculated from the image captured before the A ⁇ agglutination reaction was about 10 (dotted line in FIG. 13b).
  • the imaging is performed under the exposure conditions that the Sum Intensity is 15% to 85%.
  • the exposure time is about 150 ms to 1.8 s and the exposure condition is 1x the camera gain or the automatic exposure of the CCD camera. It became clear that it had to be done using features.
  • the imaging is performed under the exposure condition that the Sum Intensity is 45 to 65%, and in the case of the conditions of the present embodiment. It has been confirmed that it is preferable to perform under exposure conditions of exposure time of about 500 ms to 900 ms and camera gain of 1 time, or using the automatic exposure function of the CCD camera.
  • Fig. 14 shows the relationship between the Sum Intensity and the original luminance value SD and the corrected luminance value SD.
  • the correction increased the brightness value SD.
  • the Sum Intensity was a value greater than 50%, the brightness value SD was reduced by the correction but was still at a level that could be used for evaluation. It has been shown that the correction of the brightness value based on the target Sum Intensity is effective not only for equalizing the Sum Intensity value but also for increasing the brightness value SD when the Sum Intensity value is small. ..
  • ⁇ Camera gain (ISO sensitivity) 25 nM QD605A ⁇ and 25 ⁇ M A ⁇ 42 were incubated to perform A ⁇ agglutination reaction, and a fluorescence microscope image was taken under the exposure conditions of a camera gain of 1 to 64 times (ISO sensitivity of 200 to 12800).
  • An image of 8 wells taken under each exposure condition is shown in FIG. 15a, and the relationship between the camera gain and the luminance value SD of the pixel included in the region of interest of each image is shown in FIG. 15b.
  • the notation of A14 to A21 in the figure indicates the position of the well on the plate.
  • Example 2 Screening of Plant Extract Using A ⁇ Aggregation Inhibition Evaluation System Screening using A ⁇ aggregation inhibitory activity as an index was carried out using 504 species of plant extract from Hokkaido, which was distributed by Professor Kenji Kade, Hokkaido University. went.
  • the method for preparing the plant extract is as follows. From June to November 2004, 504 kinds of wild plants or cultivated plants were collected in Hokkaido. The collected plants were washed for a short time, cut into small pieces using pruning scissors, and dried with hot air at 50° C. for 24 hours. The dried plant was powdered with a mixer, and then 20 g of the dry powder was extracted with 200 mL of methanol at room temperature for 24 hours. After filtration, the methanol solution was concentrated under reduced pressure and the obtained residue was stored as a stock of crude plant extract at -20°C until use. The average yield was about 25% on a dry weight basis.
  • Each extract was dissolved in DMSO to prepare a 100 mg/ml solution, and this DMSO solution was diluted to prepare 6-fold 5-fold dilution series from 0.1 mg/ml.
  • the A ⁇ aggregation inhibitory activity was evaluated under the conditions of Example 1(3), and the EC 50 value was determined.
  • Example 3 Screening of Compound Library Using A ⁇ Aggregation Inhibition Evaluation System
  • a ⁇ aggregation inhibitory activity was used as an index using 98 kinds of aromatic low molecular weight compounds which were distributed by Associate Professor Kiminori Ohta of Showa University Screened. Each compound was dissolved in DMSO to prepare a 20 mM solution, and this DMSO solution was diluted to prepare a 5-fold 5-fold dilution series from 300 ⁇ M. Using each diluted compound solution, the A ⁇ aggregation inhibitory activity was evaluated under the same conditions as in Example 1(3), except that the automatic liquid dispensing robot system was not used, and the EC 50 value was determined. did.
  • a conventional A ⁇ aggregation inhibition evaluation method (ThT assay; method using the conventional thioflavin T (ThT); the method is Ishigaki Y. et. EC 50 values according to (see al.). According to this evaluation system, the activity was confirmed even for the substance whose activity could not be detected by the conventional ThT assay.
  • Example 4 Establishment of tau aggregation inhibitory activity evaluation system (1) Material and method -Preparation of fluorescent probe QDTau Mouse tau microtubule binding domain (MDB) fragment (SEQ ID NO: 3) was previously reported (Tokuraku K. et. al., J Biol Chem., 278(32):29609-18, 2003, The literature was prepared according to (incorporated herein by reference in its entirety). The fluorescent probe QDTau was prepared by labeling the tau MDB fragment with Qdot in the same manner as in Example 1(1).
  • MDB microtubule binding domain
  • QD-PEG-NH 2 Qdot® 605 ITKTM Amino (PEG) Quantum Dots; Q21501MP, Thermo Fisher Scientific
  • 10 ⁇ M QD-PEG-NH 2 Qdot® 605 ITKTM Amino (PEG) Quantum Dots; Q21501MP, Thermo Fisher Scientific
  • 1 mM sulfo-EMCS 22307, Pierce
  • the sulfo-EMCS bound to QD-PEG-NH 2 was treated with 62.5 ⁇ M tau MDB fragment containing 5 mM EDTA in PBS for 1 h at room temperature.
  • QD Tau was prepared by reacting.
  • DTT concentration tau MDB fragment has two cysteine residues (see Fig. 20a) that may form intramolecular disulfide bridges and inhibit aggregation, so dithiothreitol which suppresses intramolecular crosslinks. (DTT) was added to the agglutination reaction solution and its effect was investigated.
  • the conditions not particularly described are the same as those of the A ⁇ aggregation reaction of Example 1.
  • ⁇ Tau MDB fragment with 0-50 ⁇ M tau concentration , 50 nM QDTau, 10 ⁇ M heparin and 10 mM DTT were incubated to perform tau agglutination reaction, and a fluorescence microscope image was taken (upper part of FIG. 19c), and the region of interest was detected.
  • the SD of the brightness value of each included pixel was calculated (Fig. 19d).
  • the aggregates were observed from the Z-axis direction with a confocal laser scanning microscope (FIG. 19c, lower stage).
  • the concentration of tau MDB fragment of 10 ⁇ M was adopted.
  • Tau precipitation assay Tau aggregation reaction was performed by incubating 10 ⁇ M tau MDB fragment, 50 nM QDTau, 10 ⁇ M heparin and 10 mM DTT. Samples having the same composition except that DTT was not added were used for comparison. After the reaction, the mixture was centrifuged at 386,000 xg for 15 minutes at 4°C to remove the supernatant, and the pellet was resuspended in the same volume of PBS. The SDS-PAGE results of the supernatant and the resuspended pellet are shown in Figure 19e. Most of the tau MDB fragments were present in the supernatant in the absence of DTT, whereas most of the tau MDB fragments were recovered in the pellet in the presence of DTT. From the above, it was shown that most of the tau MDB fragments in the reaction solution aggregate by the agglutination reaction under the above conditions.
  • Tau agglutination reaction was carried out by incubating 10 ⁇ M tau MDB fragment, 50 nM QDTau, 10 ⁇ M heparin and 10 mM DTT at 37° C. for 1 day.
  • the A ⁇ agglutination reaction was performed by incubating 30 nM QD605 A ⁇ and 30 ⁇ M A ⁇ 42 at 37° C. for 1 day.
  • Fig. 20g shows the relationship between the reaction time and the luminance value SD of the pixels included in the region of interest in the fluorescence microscope images taken over time in both agglutination reactions. It was confirmed that the brightness value SD reached equilibrium in both tau and A ⁇ with a reaction time of about 16 hours.
  • Example 5 Verification of Tau Aggregation Inhibitory Activity Evaluation System and A ⁇ Aggregation Inhibitory Activity Evaluation System Chaperone (calreticulin (CRT), ERp57, protein disulfide isomerase (PDI)) having A ⁇ aggregation inhibitory activity as a test substance; Kitauchi, K. et. al., Biochem. Biophys. Res. Commun. 481, 227-231, 2016). CRT, ERp57 and PDI were prepared according to previous reports (Sakono M. et. al., Biochem. Biophys. Res. Commun. 452, 27-31, 2014, which is incorporated herein by reference in its entirety).
  • each chaperone has tau and A ⁇ aggregation inhibitory activity, and in particular has stronger activity against A ⁇ (FIG. 21).
  • Example 6 Evaluation of Amyloid Aggregation Inhibitory Activity Using Quantum Dots as Fluorescent Probe Quantum Dots QD-PEG-NH 2 (Qdot (registered trademark) 655 ITK (trademark) Amino (PEG) Quantum was used as a fluorescent probe instead of QDA ⁇ and QDtau. Dots; Q21521MP) was used, and the agglutination reaction of tau and A ⁇ was performed under the same conditions as in Example 4(3). The ⁇ -synuclein agglutination reaction was performed as follows.
  • Human ⁇ -synuclein (SEQ ID NO: 4) was prepared by purifying by genetically modified Escherichia coli and liquid column chromatography in the same manner as the Tau MDB fragment (from Associate Professor Taro Noguchi, Miyakonojo National College of Technology). Share). 400 ⁇ M ⁇ -synuclein monomer was stirred with a stirrer at 37° C. for 1 week in a PBS solution containing 10 mM MgCl 2 to form ⁇ -synuclein aggregates ( ⁇ -synuclein seeds) as seeds.
  • Fig. 24A shows a fluorescence microscope image before and after the reaction
  • Fig. 24B shows a brightness value SD calculated from the image. Since the distribution of ThT was biased by the aggregation of A ⁇ , and the value of the brightness value SD also increased after the reaction, it was shown that ThT can also be used as a fluorescent probe in the present amyloid aggregation inhibitory activity evaluation system.

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Abstract

La présente invention concerne : un procédé consistant à déterminer des activités inhibitrices de formation d'amyloïde de substances de test, le procédé comprenant : une étape consistant à faire réagir, en présence ou en l'absence d'une substance de test, une protéine amyloïdogène avec une sonde fluorescente capable de se lier à l'amyloïde formée par polymérisation de la protéine amyloïdogène ; une étape consistant à capturer la fluorescence des produits de réaction d'agglutination dans une condition d'exposition dans laquelle une intensité de somme calculée à partir de la valeur de luminosité de chaque pixel inclus dans une région d'intérêt dans une image de fluorescence devant être capturée est de 15 à 85 % ; une étape consistant à calculer l'écart type à partir de la valeur de luminosité de chaque pixel inclus dans la région d'intérêt dans l'image de fluorescence capturée ; et une étape consistant à comparer l'écart-type calculé dans des valeurs de luminosité en présence de la substance de test et de l'écart-type dans des valeurs de luminosité en l'absence de la substance de test, et à déterminer que la substance de test a une activité inhibitrice de formation d'amyloïde lorsque l'écart-type dans les valeurs de luminosité en présence de la substance de test est inférieur à l'écart-type dans des valeurs de luminosité en l'absence de la substance de test ; et un appareil et un programme qui peuvent être utilisés dans la mise en œuvre du procédé.
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WO2022211014A1 (fr) * 2021-03-31 2022-10-06 株式会社カネカ Procédé de mesure de protéine d'agrégation, procédé de visualisation d'agrégation et kit destiné à être utilisé dans lesdits procédés
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WO2024070962A1 (fr) * 2022-09-27 2024-04-04 株式会社カネカ Inhibiteur d'agrégation

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WO2022211014A1 (fr) * 2021-03-31 2022-10-06 株式会社カネカ Procédé de mesure de protéine d'agrégation, procédé de visualisation d'agrégation et kit destiné à être utilisé dans lesdits procédés
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