WO2021090922A1 - METHOD FOR QUICKLY AND EASILY QUANTIZING DENATURED LDL AND STIMULATIVE AGEs - Google Patents

METHOD FOR QUICKLY AND EASILY QUANTIZING DENATURED LDL AND STIMULATIVE AGEs Download PDF

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WO2021090922A1
WO2021090922A1 PCT/JP2020/041569 JP2020041569W WO2021090922A1 WO 2021090922 A1 WO2021090922 A1 WO 2021090922A1 JP 2020041569 W JP2020041569 W JP 2020041569W WO 2021090922 A1 WO2021090922 A1 WO 2021090922A1
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molecule
kit
sample
biomarker
antibody
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PCT/JP2020/041569
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French (fr)
Japanese (ja)
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町田　幸子
みゆき 倉持
俊郎 小堀
秀樹 瀬筒
謙一郎 立松
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国立研究開発法人農業・食品産業技術総合研究機構
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Priority to JP2021555126A priority Critical patent/JPWO2021090922A1/ja
Publication of WO2021090922A1 publication Critical patent/WO2021090922A1/en

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a technique for simply and quickly quantitatively detecting denatured LDL or stimulating AGEs.
  • Oxidized LDL (so-called "bad cholesterol”) is known as a risk factor for dyslipidemia, ischemic heart disease, etc.
  • Oxidized LDL is a general term for oxidatively modified LDL molecules and the degree of modification. Is also a different heterogeneous population of molecules.
  • the inventors have succeeded in developing a method for widely detecting only oxidized LDL (true bad) that triggers a disease in a living body from oxidatively modified LDL molecules (Patent Document 1).
  • the developed method requires specialized technology and equipment, and is premised on use in research institutes and inspection institutions.
  • AGEs are glycation products of proteins, but are a general term for various structures.
  • AGEs caused by glycation stress also exist in the human body, and include stimulating AGEs that can induce diabetic complications and age-related diseases (rheumatoid arthritis, Alzheimer's disease, etc.). It was. The inventors have succeeded in developing a method for detecting AGEs that trigger diseases from among AGEs having various structures.
  • the developed method is premised on use in research institutes, inspection institutions, etc., like the oxidized LDL detection method, and is based on the effect of suppressing the progression of diseases by improving lifestyle such as exercise and dietary habits, and by medication. There is a need to develop a method for quantitative and quick quantitative evaluation of disease management status.
  • the present inventors have developed a technique capable of quantifying oxidized LDL or stimulating AGEs, for example, a lateral flow assay (immunochromatography). Specifically, in the quantification technique, simple and rapid quantification of oxidized LDL or stimulating AGEs was achieved by using a recognition element modified with fluorescent nanoparticles that recognize oxidized LDL or stimulating AGEs. ..
  • the present disclosure provides, for example,: (Item 1) A device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule, which comprises a membrane for developing a sample by capillarity, wherein the membrane is: A detection unit containing a detection binder that specifically binds to a biomarker molecule or a competing molecule thereof, and a control bond that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having the ability to form a conjugate with the competing molecule.
  • the kit or device comprises a sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
  • (Item 2) The kit or device according to item 1, wherein the membrane includes the detection unit and the control unit in this order from upstream to downstream.
  • (Item 3) The kit or device according to items 1 and 1A, wherein the sample contact portion, the detection portion, and the control portion are arranged or connected to each other so that the sample permeates through a capillary phenomenon.
  • (Item 4) The device or kit according to any one of items 1 to 3, wherein the biological marker molecule is LDL or AGEs.
  • (Item 5) The device or kit according to any one of items 1 to 4, wherein the aberrant form of the biomarker molecule is denatured LDL or stimulating AGEs.
  • the detection binding agent is any one of items 1 to 6, which is an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, or an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof.
  • the device or kit described in the section. (Item 8) The device or kit according to any one of items 1 to 7, wherein the kit or device further comprises a competing molecule of the biomarker molecule.
  • the biomarker molecule is LDL, the variant of the biomarker molecule is denatured LDL, the binding molecule is CTLD14, and the detection binding agent is anti-LDL antibody, anti-modified LDL antibody or anti-ApoB antibody or them.
  • the device or kit according to any one of items 1 to 8, which is an antigen-binding fragment of. (Item 10)
  • the biomarker molecule is AGEs, the variant of the biomarker molecule is stimulating AGEs, the binding molecule is sRAGE, and the detection binding agent is an anti-BSA antibody or anti-OVA antibody or an antigen-binding fragment thereof.
  • the kit or device further comprises a competing molecule of the biomarker molecule, wherein the competing molecule is G-BSA or G-OVA.
  • the fluorescent nanoparticles are provided as a detection reagent.
  • the fluorescent nanoparticles are provided as a sample mixture in the membrane.
  • the device or kit of item 13 wherein the blood cell separator is selected from FUSION5, LF1, MF1, and VF2.
  • the CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case, the control binder is streptavidin, an anti-His antibody,
  • the device or kit of item 4 which is an anti-Myc antibody, an anti-Flag antibody, an anti-E-tag antibody, or a Strept-Tactin.
  • (Item 17) The device or kit of item 6, wherein the CTLD14 is biotinylated and the control binder is streptavidin.
  • (Item 18) The device or kit according to any one of items 1 to 17, wherein the sample is a blood sample.
  • (Item 19) The device or kit of item 18, wherein the blood sample is serum or whole blood.
  • (Item 20) A method for detecting or quantifying abnormal types of biological marker molecules. The process of providing the sample and A step of mixing the sample with a binding molecule labeled with fluorescent nanoparticles, wherein the binding molecule has the ability to form a conjugate with an aberrant form of a biomarker molecule or a competing molecule thereof.
  • a method comprising the step of adding a buffer as needed after contact.
  • the device, system or kit comprises a membrane that develops a sample by capillary action.
  • the sample contact part and the detection part containing a detection binder that specifically binds to the biomarker molecule or its competing molecule and the abnormal type of the biomarker molecule or the binding molecule having the ability to form a conjugate with the competing molecule Includes a control unit containing a control binder
  • the fluorescent nanoparticles are used by labeling the binding molecule.
  • Composition. (Item 22) A composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising a binding molecule labeled with fluorescent nanoparticles, said binding molecule of the biomarker molecule.
  • the device, system or kit comprises a membrane that develops a sample by capillary action. It includes a sample contact part, a detection part containing a detection binder that specifically binds to a biological marker molecule or a competing molecule thereof, and a control part that contains a control binder that specifically binds to the binding molecule. Composition.
  • a device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule which comprises a membrane for developing a sample by capillarity, wherein the membrane is: It includes a detection part containing a competing molecule of a biomarker molecule and a control part containing a control binder that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having a conjugate forming ability with the competing molecule.
  • the kit or device comprises a sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
  • (Item 24) 23 The kit or device according to claim 23, which has the characteristics of one or more of the above items.
  • the device, system or kit comprises a membrane that develops a sample by capillary action. Includes a sample contact part, a detection part containing a competing molecule of a biomarker molecule, and a control part containing a control binder that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having the ability to form a conjugate with the competing molecule.
  • the fluorescent nanoparticles are used by labeling the binding molecule. Composition.
  • a composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule comprising a binding molecule labeled with fluorescent nanoparticles, said binding molecule of the biomarker molecule.
  • the device, system or kit comprises a membrane that develops a sample by capillary action. It includes a sample contact part, a detection part containing a competing molecule of a biological marker molecule, and a control part containing a control binder that specifically binds to the binding molecule.
  • Composition (Item 27) The composition according to claim 25 or 26, which has the characteristics of the above one or more items.
  • (Item 28) A method for detecting or quantifying abnormal types of biological marker molecules.
  • a method comprising the step of adding a buffer as needed after contact. (Item 29) 28.
  • the method of claim 28 which has the characteristics of one or more of the above items.
  • a device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule which comprises a membrane for developing a sample by capillarity, wherein the membrane is:
  • the sample contact part and the detection part containing a detection binder that specifically binds to the biomarker molecule or its competing molecule and the abnormal type of the biomarker molecule or the binding molecule having the ability to form a conjugate with the competing molecule Includes a control unit containing a control binder
  • the kit or device comprises fluorescent nanoparticles as part of or as a separate element of the membrane.
  • the detection binding agent is any one of items 1A to 4A, which is an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, or an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof.
  • the biomarker molecule is LDL
  • the variant of the biomarker molecule is denatured LDL
  • the binding molecule is CTLD14
  • the detection binding agent is anti-LDL antibody, anti-modified LDL antibody or anti-ApoB antibody or them.
  • the device or kit according to any one of items 1A to 6A which is an antigen-binding fragment of.
  • the biomarker molecule is AGEs, the variant of the biomarker molecule is stimulating AGEs, the binding molecule is sRAGE, and the detection binding agent is an anti-BSA antibody or anti-OVA antibody or an antigen-binding fragment thereof.
  • the fluorescent nanoparticles are provided as a detection reagent.
  • (Item 10A) The device or kit according to any one of items 1A to 8A, wherein the fluorescent nanoparticles are provided as a sample mixture in the membrane.
  • (Item 11A) The device or kit according to any one of items 1A to 10A, wherein the membrane further comprises a blood cell separator.
  • (Item 12A) The device or kit of item 11A, wherein the blood cell separator is selected from FUSION5, LF1, MF1, and VF2.
  • the CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case, the control binder is streptavidin, an anti-His antibody,
  • the device or kit of item 4A which is an anti-Myc antibody, an anti-Flag antibody, an anti-E-tag antibody, or Strept-Tactin.
  • (Item 16A) The device or kit according to any one of items 1A to 15A, wherein the sample is a blood sample.
  • (Item 17A) A method for detecting or quantifying abnormal types of biological marker molecules. The process of providing the sample and A step of mixing the sample with a binding molecule labeled with fluorescent nanoparticles, wherein the binding molecule has the ability to form a conjugate with an aberrant form of a biomarker molecule or a competing molecule thereof. A step of contacting the sample contact portion of the membrane with the mixed sample in the device or kit according to any one of items 1A to 16A. A method comprising the step of adding a buffer as needed after contact.
  • the device, system or kit comprises a membrane that develops a sample by capillary action.
  • the sample contact part and the detection part containing a detection binder that specifically binds to the biomarker molecule or its competing molecule and the abnormal type of the biomarker molecule or the binding molecule having the ability to form a conjugate with the competing molecule includes a control unit containing a control binder
  • the fluorescent nanoparticles are used by labeling the binding molecule. Composition.
  • a composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule comprising a binding molecule labeled with fluorescent nanoparticles, said binding molecule of the biomarker molecule.
  • the device, system or kit comprises a membrane that develops a sample by capillary action.
  • a composition comprising a sample contact portion, a detection portion containing a detection binder that specifically binds to a biological marker molecule or a competing molecule thereof, and a control portion that contains a control binder that specifically binds to the binding molecule.
  • the present disclosure is provided a technique capable of easily and quickly quantifying oxidized LDL or stimulant AGEs associated with a disease. Therefore, the present disclosure is an oxidized LDL or irritant useful for diagnosing diseases (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer-type dementia), assessing the effectiveness of treatment, and taking preventive measures. Kits and methods for detecting AGEs, as well as substrates that can be used for it are provided.
  • FIG. 1 shows an outline of single chain antibody production.
  • FIG. 2 shows the results of detection of oxidized LDL by the lateral flow (immunochromatography) assay.
  • FIG. 3 shows the results of measuring the fluorescence intensity of each spot of the strip in FIG.
  • FIG. 4 shows the results of detection of added oxidized LDL in serum by immunochromatography assay.
  • FIG. 5 shows a schematic diagram of the principle of detection of denatured LDL by lateral flow assay.
  • FIG. 6 shows the correlation between the oxidized LDL concentration in the serum of hyperlipidemic patients and the management status judged from the LDL, HDL, and TG concentrations of dyslipidemia patients.
  • abnormalities are based on the classification (left) based on whether the LDL, HDL, and TG values of patients with dyslipidemia are within the control target values or are judged to be abnormal.
  • Patient sera were classified into levels 0 to 4 (lower right), and the range of oxidized LDL concentration for each level was arranged (upper right) with reference to the number of items classified as.
  • FIG. 7 shows a comparison of oxidized LDL concentrations by an immunochromatographic assay using sera of hyperlipidemic patients classified in FIG.
  • FIG. 8 shows the results of detection of oxidized LDL added to whole blood samples by immunochromatography assay.
  • FIG. 10 shows a schematic diagram of the principle of detection of stimulating AGEs by the lateral flow assay.
  • FIG. 11 shows the results of a lateral flow assay using a model sample.
  • FIG. 12 shows the results of detection of AGEs in serum by the lateral flow assay. The image on the left of FIG. 12 shows the image acquired by the image analyzer, and the graph on the right shows the fluorescence intensity of each spot.
  • FIG. 13 shows the results of detection of AGEs in whole blood by the lateral flow assay. The image on the left of FIG.
  • FIG. 13 shows an image acquired by an image analyzer, and the graph on the right shows the fluorescence intensity of each spot.
  • FIG. 14 shows the results of detection of AGEs in whole blood by the lateral flow assay. The image on the left of FIG. 14 shows the image acquired by the image analyzer, and the graph on the right shows the fluorescence intensity of each spot.
  • FIG. 15 shows the results of detection of AGEs in the serum of NASH patients by the lateral flow assay. The image on the left of FIG. 15 shows the image acquired by the image analyzer, and the graph on the right shows the fluorescence intensity of each spot.
  • FIG. 16 shows the results of detection of AGEs in the serum of diabetic complication patients by the lateral flow assay. The image on the left of FIG.
  • FIG. 16 shows an image acquired by an image analyzer, and the graph on the right shows the fluorescence intensity of each spot.
  • FIG. 17 shows a schematic diagram of a detection system in which a competing molecule (CML saccharified BSA) is applied to a detection unit (test spot).
  • CML saccharified BSA CML saccharified BSA
  • system refers to any system for performing detection, predictive diagnosis, pre-diagnosis, diagnosis, etc., and generally consists of one or more components, and if there are a plurality of components, those.
  • the elements of are acting and related to each other, and refer to a system that satisfies the three conditions of exhibiting harmonious behavior and function as a whole.
  • the system can be in any form such as a device, device, composition, diagnostic agent.
  • the system can be, for example, from a large-scale system with a measuring device, to a system with chromatography, a kit / combination utilizing an immune reaction, a composition containing an antibody (ie, an in vitro drug containing a monoclonal antibody of a marker). It is understood to include certain diagnostic agents) and the like.
  • the term "device” refers to any device for performing detection, predictive diagnosis, pre-diagnosis, diagnosis, etc., and is composed of one or a plurality of components, and if there are a plurality of components, those components are usually used. Are often in contact with each other and are used to refer to devices, appliances, tools, and objects used for a specific purpose in general, and are not construed as being limited to those having mechanical or electrical action. Includes at least one element that is operably linked to each other to enable normal purpose (eg, examination, detection, diagnosis, etc.).
  • kits refers to a unit that is usually divided into two or more compartments and is provided with parts to be provided (eg, membranes, devices, reagents, etc.). If multiple reagents or devices are provided independently, it may be convenient to provide them as this kit. Such kits preferably include instructions or instructions describing how to use the provided parts (eg, membranes or devices) or how to use the reagents. Is advantageous.
  • the "instruction” describes the method for using the present disclosure to the user.
  • This instruction contains language that directs how to use this disclosure. If necessary, this instruction may be provided by the regulatory agency of the country in which this disclosure is implemented (eg, Ministry of Health, Labor and Welfare or Ministry of Agriculture, Forestry and Fisheries in Japan, Food and Drug Administration (FDA), Department of Agriculture (USDA) in the United States. ) Etc.), and it is clearly stated that it has been approved by the regulatory agency. Instructions may be provided in paper media, but are not limited to, and may also be provided in the form of, for example, electronic media (eg, homepages, e-mails, SNS, simple messages, etc. provided on the Internet).
  • electronic media eg, homepages, e-mails, SNS, simple messages, etc. provided on the Internet.
  • the "fluorescent nanoparticles” are nanoparticles of nano-sized particles capable of emitting fluorescence having sufficient intensity to detect a target biological substance.
  • the fluorescent nanoparticles quantum dots (semiconductor nanoparticles) and fluorescent substance-integrated nanoparticles are preferably used.
  • quantum dots semiconductor nanoparticles containing a group II-VI compound, a group III-V compound, or a group IV element are used.
  • CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, Ge and the like can be mentioned.
  • Fluorescent substance-accumulated nanoparticles are based on particles made of organic or inorganic substances, and a plurality of fluorescent substances (for example, the quantum dots, fluorescent dyes, etc.) are contained therein and / or adsorbed on the surface thereof. It is a nano-sized particle having a structure.
  • the mother body and the fluorescent substance have substituents or sites having opposite charges and electrostatically interact with each other.
  • the fluorescent substance integrated nanoparticles quantum dot integrated nanoparticles, fluorescent dye integrated nanoparticles and the like are used.
  • the organic substances are resins generally classified as thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin; styrene resin, acrylic resin, and acrylonitrile.
  • Resins generally classified as thermoplastic resins such as resins, AS resins (acrylonitrile-styrene copolymers), ASA resins (acrylonitrile-styrene-methyl acrylate copolymers); other resins such as polylactic acid; polysaccharides Can be exemplified.
  • the inorganic substance in the mother body include silica and glass.
  • Quantum dot integrated nanoparticles have a structure in which the quantum dots are contained in the parent body and / or adsorbed on the surface thereof.
  • the quantum dots may or may not be chemically bonded to the mother body itself as long as they are dispersed inside the mother body.
  • Fluorescent dye-accumulated nanoparticles have a structure in which a fluorescent dye is contained in the mother body and / or is adsorbed on the surface thereof.
  • the fluorescent dye include rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic ring-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, and pyromesene-based dye molecules.
  • the fluorescent dye include Alexa Fluor (registered trademark, Invigen) dye molecule, BODIPY (registered trademark, Invigen) dye molecule, Cy (registered trademark, GE Healthcare) dye molecule, and HiLite (registered).
  • Alexa Fluor 514 Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor6 Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 601 / / 665 (manufactured by Invitrogen), methoxykumarin, kumarin 6, kumarin 7, sulfokumarin 6, sulfokumarin 7, eodin, NBD, pyrene, Cy5, Cy5.5, Cy7, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluorescein 594, HiLite Fluor 647, HiLyte
  • fluorescent dyes may be used alone or in combination of two or more. It should be noted that such dye molecules are generically named based on the main structure (skeleton) in the compound or a registered trademark, and the range of fluorescent dyes belonging to each requires excessive trial and error by those skilled in the art. It can be grasped properly without any problem.
  • the fluorescent dye When the fluorescent dye is encapsulated in the mother body, the fluorescent dye may or may not be chemically bonded to the mother body itself as long as it is dispersed inside the mother body.
  • a "biological marker molecule” is a substance that serves as a marker for tracking whether a condition (eg, disease, disorder, etc.) is present or at risk. .. Examples of such markers include genes, gene products, metabolites, enzymes and the like.
  • examples of the biomarker molecule include LDL, AGEs, and analogs thereof.
  • Some biological marker molecules have different levels or morphologies in the healthy state and non-healthy states, and are characteristically found in other than the healthy state), which are particularly referred to herein as "abnormal forms of the biological marker molecule".
  • Abnormal forms of biomarker molecules can be detected in the present invention because they are particularly strongly associated with disease. Abnormal forms of biomarker molecules are often modifications of biomarkers that are not normally observed or are rarely observed in healthy individuals. Abnormal forms of biomarker molecules include denatured LDL and stimulant AGEs.
  • the "competitive molecule” means a binding molecule that binds to a certain object and a molecule that competes and binds to a certain object. Since the binding of a competing molecule to a certain object competes with the binding molecule, the amount of the binding molecule bound to the competing molecule decreases when the competing molecule exists. Therefore, by using the competing molecule, the binding molecule or bond (for example, Conju) can be used. The amount of gate formation) can be measured indirectly.
  • conjugate means that a certain object and another entity are combined and integrated, and the ability thereof is referred to as “conjugate forming ability”.
  • CLTD14 corresponds to denatured LDL
  • sRAGE corresponds to stimulating AGEs
  • other molecules can be appropriately specified by those skilled in the art.
  • a "detecting binder” is a biomarker molecule or anomalous form thereof to be detected at a detection site (eg, test spot or line) of a membrane in the devices or kits of the present disclosure. , Or a molecule that specifically binds to these competing molecules (eg, anti-LDL antibody, anti-modified LDL antibody or anti-ApoB antibody or antigen-binding fragment thereof, or anti-BSA antibody or anti-OVA antibody or antigen-binding fragment thereof, etc. ).
  • control binder is an atypical form of a biomarker molecule and the ability to form a conjugate in a control section (eg, control spot or line) of a membrane in a device or kit of the present disclosure.
  • a control section eg, control spot or line
  • a binding molecule that has the ability to form a conjugate with an atypical competing molecule of a binding molecule or a biological marker molecule.
  • control binding agent is a binding molecule (eg, CTLD14) capable of forming a conjugate with a molecule that specifically binds to an aberrant form of the biomarker molecule (eg, modified LDL), or It can be a binding molecule (sRAGE) capable of forming a conjugate with a competing molecule (eg, G-BSA) that competes with the biomarker molecule (eg, stimulating AGEs), and an abnormal type of the biomarker molecule has been identified.
  • a binding molecule eg, CTLD14
  • sRAGE binding molecule capable of forming a conjugate with a competing molecule (eg, G-BSA) that competes with the biomarker molecule (eg, stimulating AGEs)
  • those skilled in the art may appropriately bind to a binding molecule having the ability to form a conjugate with the molecule (for example, CTLD14 in the case of modified LDL) (for example, anti-LDL antibody in the case of modified LDL, anti-modified LDL).
  • a competing molecule thereof eg, G-BSA or G-OVA in the case of stimulating AGEs
  • an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof can be identified or produced, and these can be used as a control binding agent.
  • membrane refers to a solid phase that is a porous or non-porous water-insoluble material, preferably one that at least partially has a material capable of binding or retaining biomolecules. possible.
  • the membrane used in the present specification is preferably capable of developing a sample by capillarity, and may contain at least a part of a material for realizing them.
  • cellulose examples include, for example, cellulose, polysaccharides such as Sephadex TM, glass, polyacryloyl morpholide, silica, controlled pore glass (CPG), polystyrene, polystyrene / Polyethylene such as latex, ultra-high molecular weight polyethylene (UPE), polyamide, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE; Teflon®), carboxyl-modified Teflon®, nylon, nitrocellulose And can be composed of metals and alloys such as gold, platinum and palladium.
  • Membranes are usually charged and bind to organic materials such as proteins. Membranes significantly improve various analytical processes by making the process quantitative.
  • modified LDL is also referred to as “LDL modified product” and “modified LDL” (these are used interchangeably), and LDL is an active oxygen species, oxidative enzyme, Fe3 + in the body. It is any LDL-modified product having various molecular modifications generated by contact with such as, or by cell-dependent chemical changes caused by vascular endothelial cells, macrophages, and the like.
  • Typical LDL modifications existing in the living body include oxidized LDL (referred to as OxLDL in the present specification, and for example, fully oxidized LDL (also referred to as fu OxLDL in the present specification) and partially oxidized LDL (the present specification).
  • oxidized LDL shows abnormal values, arteriosclerosis, ischemic heart disease (myocardial infarction, angina, etc.), cerebrovascular accident (cerebral infarction, cerebral bleeding, submucosal bleeding, transient ischemic attack, etc.), Diseases such as aortic aneurysm, renal infarction, and hyperlipidemia are expected, but not limited to these (see “Today's Clinical Examination 2007-2008” Publisher Nanedo Co., Ltd.).
  • MDA-LDL normal range: 10 to 80 ⁇ L
  • oxidized phosphatidylcholine normal range: 8.4 U / mL to 17.6 U / mL
  • CTLD molecule is understood to include CTLD-like polypeptides as well as any complex thereof. Therefore, it is understood that the CTLD molecule also includes the LOX-1 total length, the LOX-1 extracellular space total length (S61 to Q273), CTLD14 (129-143), CTLD (143-273), and the like.
  • CTLD14 and PR-CTLD14 are: (1) a polypeptide consisting of the nucleic acid sequence set forth in SEQ ID NO: 2; (2) the amino acid sequence set forth in SEQ ID NO: 2 above.
  • the 104th and 121st amino acids are polypeptides that retain the corresponding amino acid in SEQ ID NO: 2 and contain an amino acid sequence exhibiting the activity of native LOX-1; (9) in the nucleic acid sequence shown in SEQ ID NO: 1 above.
  • a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having one or several substitutions, additions and / or deletions and exhibiting the activity of native LOX-1; (10) Nucleic acid set forth in SEQ ID NO: 1 above.
  • the above identity or homology is calculated using default parameters using BLAST (NCBI BLAST 2.9 (issued March 11, 2019)), which is a tool for sequence analysis. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of one of ordinary skill in the art.
  • AGEs are glycation products of proteins and have various structures. It is a general term for the body. It occurs in the process of processing foods and is important for improving the taste, but it is also produced in the living body, and a part of it induces dysfunction in the living body and triggers age-related diseases. It is also known to be involved in the onset and progression of diabetic angiopathy known as vascular complications, which is the main cause of impairing the quality of life of diabetic patients.
  • diabetic retinopathy Damage to the eyes, nerves, and kidneys due to vascular complications is called diabetic retinopathy, neuropathy, and nephropathy (total of the three major complications), and is a pathological condition characteristic of diabetic patients.
  • Reducing sugars such as glucose react non-enzymatically with amino groups of proteins and amino acids to form glycation products such as Schiff bases or Amadori rearrangement compounds.
  • the reaction up to this point is reversible and is called the early reaction.
  • advanced glycation end products are formed through complicated and irreversible reactions such as condensation, cleavage, and crosslink formation. Such a series of reactions is called a saccharification reaction.
  • AGEs are also a general term for structures produced through such a process.
  • Examples of the AGEs structure present in the living body include, but are not limited to, carboxymethyl lysine (CML), carboxyethyl lysine (CEL), pentosidine, pyrarin, imidazoline, methylglyoxal, and crosulin.
  • CML carboxymethyl lysine
  • CEL carboxyethyl lysine
  • pentosidine pyrarin
  • imidazoline methylglyoxal
  • crosulin crosulin.
  • the product in which albumin, immunoglobulin, ovalbumin and the like present in plasma undergo the above-mentioned saccharification is also AGE, and is widely used in experimental systems as AGE.
  • BSA bovine serum albumin
  • R-AGE BSA that has been glycated with ribose
  • F-AGE BSA that has been treated with fructose
  • G- AGE BSA saccharified with glucose
  • Hemoglobin A1c which is used as an index of glycemic control, is an Amadori rearrangement compound, but is included in AGEs.
  • any protein can be converted to AGEs.
  • CML albumin and CEL albumin included in AGEs are both AGEs in which albumin is glycated.
  • Such an AGEs production reaction can occur in the circulating blood, extracellular matrix, or intracellular in vivo.
  • AGEs present in the blood vessels of diabetic patients include: fluorescent and cross-linked structures (pentosidine, crosulin, etc.) and non-fluorescent and non-cross-linked (carboxymethyl lysine, pyrarin, methylglyoxal (MG) -imidazolone, etc.) )
  • AGEs show abnormal values, diseases such as microangiopathy (nephropathy, retinopathy, neuropathy, etc.) and macroangiopathy (ischemic heart disease, cerebrovascular disease, arteriosclerosis obliterans, etc.) are expected.
  • the reference substances are pyrarin (normal range: less than 23 pmol / mL in plasma) and pentocidin (normal range: 0.00915 to 0.0431 ⁇ g / mL in plasma (as measured by ELISA)). Etc. are used (see “Today's Clinical Examination 2007-2008” Publisher, Nanedo Co., Ltd.).
  • stimulated advanced glycation end products or “stimulated AGEs (stimulated AGEs)” refer to AGEs that are highly related to diseases and have a property of strongly binding to sRAGE. .. Previously, it was thought that glycation by glucose in blood was the main cause, but glycation by glucose takes a long time, and it has begun to be suggested that glucose glycated AGEs are less irritating to the living body.
  • AGEs molecule means any molecule contained in the above-mentioned AGE.
  • AGEs include Lys-AGE (glutaraldehyde-modified lysine-modified AGE), glucose-modified AGE (G-AGE), ribose-modified AGE (R-AGE), fructose-modified AGE (F-AGE), or a variant thereof.
  • Lys-AGE glutaraldehyde-modified lysine-modified AGE
  • G-AGE glucose-modified AGE
  • R-AGE ribose-modified AGE
  • F-AGE fructose-modified AGE
  • Complexes thereof can be mentioned, but are not limited to them.
  • the "molecule exhibiting AGEs-like activity” refers to a molecule having at least one of the above-mentioned activities of AGEs (referred to as “AGEs-like activity” in the present specification).
  • AGEs-like activity examples include, but are not limited to, binding activity to RAGE (ligand activity).
  • Receptor for AGE is also referred to as RAGE, and is (1) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; (2) the above-mentioned SEQ ID NO: 4 A polypeptide containing an amino acid sequence containing one or several amino acid substitutions, additions and / or deletions in the nucleic acid sequence shown in the above, and exhibiting the activity of natural RAGE; (3) Nucleic acid shown in SEQ ID NO: 4 above. A polypeptide containing an amino acid sequence having at least 90% sequence identity with the sequence and exhibiting the activity of natural RAGE; (4) Nucleic acid having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 4 above.
  • a polypeptide containing a sequence and exhibiting the activity of native RAGE (5) a polypeptide containing an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO: 3; (6) in the nucleic acid sequence shown in SEQ ID NO: 3 above.
  • a polypeptide containing an amino acid sequence encoded by a nucleic acid molecule that hybridizes with a complementary nucleic acid sequence and a nucleic acid molecule that hybridizes under stringent conditions, and exhibits the activity of natural RAGE (7) Nucleic acid shown in SEQ ID NO: 3 above.
  • RAGE is also a type I membrane protein with a molecular weight of approximately 35 kDa (a complete RAGE with sugar chain modification has a molecular weight of 55 kDa), which was identified in bovine lung in 1992 and belongs to the immunoglobulin superfamily that binds to AGE. ..
  • the extracellular domain of RAGEs has a structure in which one V-type immunoglobulin domain is followed by two C-type immunoglobulin domains (C1 region and C2 region), which have three immunoglobulin fold structures. There is.
  • RAGE also contains a transmembrane domain and a 43 amino acid cytoplasmic domain.
  • RAGE interacts with various classes of ligands (AGEs, S100 / calgranulin, amphoterin and amyloid- ⁇ peptide).
  • the V domain is an essential site for ligand binding and the cytoplasmic domain is essential for RAGE-mediated intracellular signaling.
  • the mutant RAGE-surfactant complex of the present disclosure is at positions 38, 99, 144, 208, 259 and 301 in the amino acid sequence of SEQ ID NO: 6. It preferably retains the cysteine residue corresponding to the position.
  • RAGE is expressed only at low levels in normal tissues and the vascular system. However, this receptor is upregulated where the ligand accumulates. RAGE expression is increased in endothelial cells, smooth muscle cells, pericytes, renal mesangial cells and infiltrating mononuclear phagocytes in the diabetic vasculature. In addition, the expression of RAGE is also increased in pathological sites such as arteriosclerotic lesions in which AGEs are accumulated.
  • AGEs-RAGE interactions alter important cellular properties in vascular homeostasis. For example, after RAGE binds to AGEs, vascular endothelial cells increase the expression of VCAM-1, tissue factor, and IL-6, as well as their permeability to macromolecules. In mononuclear phagocytes, RAGE activates cytokine and growth factor expression and induces cell migration in response to soluble AGEs, whereas haptotaxis occurs with fixed ligands.
  • RAGE ligand recognition region or “sRAGE (Soluble Receptor Advanced Glycation End products)” is used interchangeably and refers to a region recognized by the RAGE ligand. Specifically, the sRAGE or RAGE ligand recognition region refers to all or part of the extracellular space of RAGE.
  • the sRAGE is typically composed of, but not limited to, positions 22-332 of SEQ ID NO: 6 or SEQ ID NO: 4.
  • RAGE-like polypeptide means “RAGE8", “mRAGE8”, “RAGE1”, “mRAGE1”, “RAGE2”, “mRAGE2”, “RAGE3”, “mRAGE3”. , “RAGE4", “mRAGE4", “RAGE7”, “mRAGE7”, “RAGE143”, “mRAGE143”, “RAGE223”, “mRAGE223”, “RAGE226” and “mRAGE226” or their variants. Including the body. These explanations are disclosed in Japanese Patent Application Laid-Open No. 2013-209330 and the like, and the contents thereof are appropriately incorporated herein by reference.
  • RAGE molecule is understood to include RAGE-like polypeptides as well as any complex thereof. Therefore, it is understood that the RAGE molecule includes RAGE-like polypeptides and the like, for example, RAGE (overall length), RAGE extracellular region (position 22-332 of SEQ ID NO: 4), RAGE143, RAGE223, RAGE226 and the like. ..
  • the RAGE molecule also includes a RAGE (mini RAGE) lacking a whole domain or a part of a certain domain among the three domains constituting the RAGE.
  • the mini-RAGE also includes a mini-RAGE of a RAGE-like polypeptide.
  • the molecule containing the "RAGE ligand recognition region” is a "RAGE molecule” other than the full-length RAGE (including the “RAGE-like polypeptide"), for example, “RAGE8", “mRAGE8", “RAGE1”. , “MRAGE1”, “RAGE2”, “mRAGE2”, “RAGE3”, “mRAGE3”, “RAGE4", “mRAGE4", “RAGE7”, “mRAGE7”, “RAGE143”, “mRAGE143”, “RAGE223”, Examples thereof include “mRAGE223", “RAGE226” and “mRAGE226", and the RAGE extracellular region (position 22-332 of SEQ ID NO: 4).
  • the RAGE-like polypeptide may contain an unnatural amino acid, an amino acid analog, an amino acid derivative, or the like as long as the activity of the natural RAGE is retained.
  • a "ligand" is a specific receptor or binding partner to a family of receptors.
  • the ligand can be an endogenous ligand for the receptor or, instead, a synthetic ligand for the receptor such as a drug, drug candidate, or pharmacological means.
  • antibody is broadly referred to as a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a chimeric antibody, and an anti-idiotype antibody, and a functional fragment thereof (for example, F (ab') 2 and the like. Fab fragments), as well as conjugates or functional equivalents produced by other recombination (eg, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies). , Single chain antibody (scFV), diabody, sc (Fv) 2 (single chain (Fv) 2 ), scFv-Fc).
  • antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase and the like. Further, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase and the like.
  • an antibody refers to a full-length antibody (eg, polyclonal antibody, monoclonal antibody, etc.), and the others may be referred to as a variant or an antigen-binding fragment.
  • the antibody used in the present disclosure may bind to its target, and its origin, type, shape, etc. are not limited. Specifically, it can be produced based on known antibodies such as non-human animal antibodies (for example, mouse antibody, rat antibody, camel antibody), human antibody, chimeric antibody, and humanized antibody. Single chain antibodies are used in the present disclosure.
  • the binding of the antibody to the target is preferably a discriminative or specific binding.
  • the variant of the antibody may be a combination of the antibody and various molecules such as polyethylene glycol. Variants of the antibody can be obtained by chemically modifying the antibody using known techniques.
  • single chain antibody is also referred to as “scFv (single chain Fv)", and the variable region regions ( VH and VL ) of the heavy and light chains of the antibody are linked with appropriate linker peptides. Corresponds to the one.
  • a single-chain antibody protein can be expressed by constructing such a construct at the gene level and introducing it into Escherichia coli using a protein expression vector.
  • fragment refers to a polypeptide or polynucleotide having a sequence length from 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n).
  • the length of the fragment can be appropriately changed according to its purpose. For example, in the case of a polypeptide, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, and so on. Amino acids such as 15, 20, 25, 30, 40, 50, and above are mentioned, and lengths represented by integers not specifically listed here (eg, 11) are also suitable as lower limits. possible.
  • a length represented by a non-integer may also be a suitable lower bound.
  • the lengths of polypeptides and polynucleotides can be expressed by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute and may have an upper limit as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include several above and below the number (or, for example, 10% above and below).
  • the length of a fragment useful herein can be determined by whether at least one of the functions of the full-length protein on which the fragment is based is retained.
  • the term "homology" of a gene means the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether or not the two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization under stringent conditions.
  • the DNA sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%. , 95%, 96%, 97%, 98% or 99%, the genes are homologous.
  • Amino acids can be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one-letter code.
  • comparison of similarity, identity and homology of amino acid sequence and base sequence is calculated using default parameters using BLAST, which is a tool for sequence analysis.
  • the identity search can be performed using, for example, NCBI's BLAST 2.9 (issued on March 11, 2019).
  • the value of identity in the present specification usually refers to the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value.
  • the term "modified” means a substance that has been partially modified from the original polypeptide or substance such as a polynucleotide. Such variants include substitution variants, addition variants, deletion variants, truncated variants, allelic variants and the like.
  • An allele is a genetic variant that belongs to the same locus and is distinguished from each other. Therefore, the "allele variant” refers to a variant having an allele relationship with a certain gene.
  • a “species homolog or homolog” is, within a species, homologous (preferably 60% or more, more preferably 80% or more,) at the amino acid or nucleotide level with a gene. Those having 85% or more, 90% or more, 95% or more homology).
  • ortholog is also called an orthologous gene, and refers to a gene derived from speciation from a common ancestor having two genes.
  • the human and mouse ⁇ -hemoglobin genes are orthologs, while the human ⁇ -hemoglobin and ⁇ -hemoglobin genes are paralogs (genes generated by gene duplication). .. Since orthologs are useful for estimating molecular phylogenetic trees, orthologs may also be useful in the present disclosure.
  • a conservatively modified variant is a nucleic acid that encodes the same or essentially the same amino acid sequence, and if the nucleic acid does not encode an amino acid sequence, it is essentially the same. Refers to an array.
  • base sequence modification methods include cleavage with restriction enzymes, ligation by treatment with DNA polymerase, Klenow fragment, DNA ligase, etc., and site-specific base substitution method using synthetic oligonucleotides (specification).
  • nucleic acid sequences herein that encode a polypeptide also describe all possible silent mutations of that nucleic acid.
  • each codon in a nucleic acid except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan
  • TGG which is usually the only codon for tryptophan
  • One amino acid can be replaced by another amino acid in a protein structure, such as the binding site of a ligand molecule, without a significant reduction or loss of the ability to interact. It is the ability and nature of a protein to interact that defines the biological function of a protein. Thus, substitutions of a particular amino acid can be made at the amino acid sequence or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after substitution. Thus, various modifications can be made in the peptides disclosed herein or in the corresponding DNA encoding the peptides, without any apparent loss of biological usefulness.
  • nucleic acids can be obtained by a well-known PCR method and can also be chemically synthesized. For example, a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.
  • Amino acid hydrophobicity index can be taken into account when designing modifications as described above.
  • the importance of the hydrophobic amino acid index in imparting interactive biological functions in proteins is generally recognized in the art (Kyte.J and Dollittle, LFJ. Mol.Biol.157 (Kyte.J and Doolittle, LFJ. Mol.Biol.157 ( 1): 105-132, 1982).
  • the hydrophobic nature of amino acids contributes to the secondary structure of the protein produced, which in turn defines the interaction of that protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
  • Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties.
  • hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such substitutions of amino acids based on hydrophobicity are efficient.
  • the following hydrophilicity indices are assigned to amino acid residues as described in US Pat. No. 4,554,101: arginine (+3.0); lysine (+3.0); aspartic acid (+3.
  • an amino acid can be replaced by another that has a similar hydrophilicity index and can still give a bioisostere.
  • the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • conservative substitution refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced is similar to that of the original amino acid in the amino acid substitution as described above.
  • conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and aspartic acid; and valine, leucine, and isoleucine, However, it is not limited to these.
  • amino acid additions, deletions, or modifications can also be made herein to make functionally equivalent polypeptides.
  • Amino acid substitution refers to substituting one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids of the original peptide.
  • the addition of amino acids means the addition of one or more amino acids, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids to the original peptide chain.
  • Amino acid deletion refers to the deletion of one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids from the original peptide.
  • Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, phosphorylation, hydroxylation, acylation (eg, acetylation) and the like.
  • the amino acid substituted or added may be a natural amino acid, an unnatural amino acid, or an amino acid analog. Natural amino acids are preferred.
  • substitution, addition and / or deletion of a polypeptide or polynucleotide means that the original polypeptide or polynucleotide is an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively. , To be replaced, to be added, or to be removed. Techniques for such substitutions, additions and / or deletions are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. These changes in the reference nucleic acid molecule or polypeptide can occur at the 5'end or 3'end of the nucleic acid molecule, as long as the desired function (eg, RAGE recognition) is retained.
  • desired function eg, RAGE recognition
  • substitutions, additions or deletions may be any number as long as it is one or more, and such a number may be increased as long as the desired function is retained in the variant having the substitution, addition or deletion. be able to.
  • such numbers can be one or several, and preferably within 20%, within 15%, within 10%, within 5%, or below 150, below 100, of the total length. It can be 50 or less, 25 or less, and so on.
  • tag sequence is used to bind a substance for selecting a molecule by a specific recognition mechanism such as a receptor-ligand, more specifically, a specific substance.
  • a substance that acts as a binding partner of for example, having a relationship such as biotin-avidin and biotin-streptavidin. Therefore, for example, a specific substance to which the tag sequence is bound can be selected by contacting the base material to which the binding partner of the tag sequence is bound.
  • tag sequences are well known in the art. Typical tag sequences include, but are not limited to, myc tags, His tags, HA, Avi tags, and the like.
  • protein protein
  • polypeptide oligopeptide
  • peptide refers to a polymer of amino acids of any length.
  • the polymer may be linear, branched or cyclic.
  • the amino acid may be natural or non-natural, or may be a modified amino acid.
  • the term may also include those assembled into a complex of multiple polypeptide chains.
  • the term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component).
  • the definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids), peptide-like compounds (eg, peptoids) and other modifications known in the art. Will be done.
  • amino acid may be natural or non-natural as long as it satisfies the object of the present disclosure.
  • amino acid derivative or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid.
  • amino acid derivatives and amino acid analogs are well known in the art. It is understood herein that amino acid derivatives and amino acid analogs can be used as alternatives as long as they can provide the same biological functions as amino acids.
  • natural amino acid means the L-isomer of a natural amino acid.
  • Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, aspartic acid, glutamic acid, glutamine, ⁇ -carboxyglutamic acid, arginine, ornitine. , And lysine. Unless otherwise specified, all amino acids referred to herein are L-form, but forms using D-form amino acids are also within the scope of the present disclosure. As used herein, the term "unnatural amino acid" means an amino acid that is not normally found in proteins.
  • unnatural amino acids examples include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, D- or L-form of homoarginine and D-phenylalanine.
  • amino acid analog refers to a molecule that is not an amino acid but is similar in physical properties and / or function to an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like.
  • Amino acid mimetics are compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.
  • Amino acids can be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one-letter code.
  • polynucleotide refers to a polymer of nucleotides of arbitrary length.
  • the term also includes “oligonucleotide derivatives" or “polynucleotide derivatives”.
  • oligonucleotide derivative refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual bond between nucleotides, and is used interchangeably.
  • such an oligonucleotide includes, for example, 2'-O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphate diester bond in an oligonucleotide is converted into a phosphorothioate bond, and a phosphate diester bond in an oligonucleotide.
  • oligonucleotide derivatives converted to N3'-P5'phosphoroamidate bond an oligonucleotide derivative in which ribose and phosphate diester bond in the oligonucleotide are converted into peptide nucleic acid bond, and uracil in the oligonucleotide is C- 5 Oligonucleotide derivatives substituted with propynyl uracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced with C-5 thiazole uracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced with C-5 propynyl citosine, oligonucleotides Oligonucleotide derivatives in which cytosine in nucleotides is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which
  • nucleic acid sequences are also conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as are the explicitly indicated sequences. Is intended to be included.
  • the degenerate codon substituent creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue.
  • nucleotide may be natural or non-natural.
  • a “nucleotide derivative” or “nucleotide analog” is one that is different from the naturally occurring nucleotide but has the same function as the original nucleotide.
  • Such nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, peptide-type nucleic acids (PNAs). Not done.
  • nucleic acid is also used interchangeably with genes, cDNAs, mRNAs, oligonucleotides, and polynucleotides. Specific nucleic acid sequences also include “splice variants". Similarly, a particular protein encoded by a nucleic acid implicitly includes any protein encoded by a splice variant of that nucleic acid. As the name suggests, "splice variants" are the product of alternative splicing of genes. After transcription, the first nucleic acid transcript can be spliced so that different (different) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Another polypeptide derived from the same nucleic acid by over-read transcription is also included in this definition. Any product of the splicing reaction, including recombinant splice products, is included in this definition.
  • the term “gene” refers to a factor that defines a genetic trait. It is usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that influences its expression is called a regulatory gene. As used herein, “gene” may refer to "polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein” “polypeptide”, “oligopeptide” and “peptide”.
  • PBS phosphate buffered saline
  • PBS (+) means containing calcium ion and magnesium ion
  • PBS (-) means calcium ion.
  • magnesium ion-free but herein, “PBS” shall mean “PBS (-)” unless explicitly stated.
  • Dulbecco's PBS ( ⁇ ) can be typically used.
  • the composition of Dulbecco's PBS ( ⁇ ) is NaCl 8 g, KCl 0.2 g, Na 2 HPO 4 1.15 g, KH 2 PO 4 0.2 g / L, (pH 7.4).
  • the term "receptor” is a biological structure comprising one or more binding domains that reversibly and specifically complex with one or more ligands.
  • this complex has a biological structure.
  • Receptors are either completely outside the cell (extracellular receptors), inside the cell membrane (but directing parts of the receptors to the outside environment and cytosol), or completely inside the cell (intracellular). Can be present in the receptor). They can also function independently of the cell.
  • Receptors in the cell membrane allow cells to communicate (eg, signal transduce) with space outside their boundaries and to function in the transport of molecules and ions inside and outside the cell.
  • the receptor may be the full length of the receptor or a fragment of the receptor.
  • the term "antigen-antibody reaction” is used in the broadest sense used in the art, and in particular, refers to a reaction based on a specific binding between an antigen and an antibody. Reagents and methods for detecting and quantifying antigens in a sample by using the immunoblot (Western blot) format as the detection system are also provided.
  • silk moth means silk moth (silkmoth) in the usual meaning, and is considered to be a kind of insect belonging to the order Lepidoptera (Lepidoptera) and Bombyx mori.
  • the official Japanese name is the silk moth (scientific name: Bombyx mori), which is the name of this larva, but generally also refers to this species in general. It feeds on mulberry and produces silk to make pupal cocoons.
  • the silk moth is also called a domestic silk moth and is not an insect that lives in the wild. The ancestor of the silk moth is thought to be the Bombyx mandarina, which inhabits East Asia.
  • the silk moth includes the mulberry.
  • the term "organism that imparts sugar chains similar to silk moth” refers to an organism that has the ability to add sugar chains similar to silk moth, such as a gene encoding an enzyme that adds sugar chains similar to silk moth. Can include transgenic organisms and the like.
  • the "silk gland” is a pair of left and right organs existing in the body of a mature silk moth, and a large amount of protein (amino acid) ingested from mulberry leaves is combined with two types of silk proteins (fibroin and sericin). Means an organ that changes to.
  • the silk glands are paired on the left and right, and secrete liquid silk, which is the raw material for eyebrows.
  • the silk gland is divided into three parts: a posterior silk gland, a middle silk gland, and an anterior silk gland.
  • any silk gland can be used for synthesis, but in consideration of post-synthesis handling, the posterior silk gland and the middle silk gland are usually used, and the middle silk gland is preferably used. It is not limited. In addition, it can be expressed in the whole body and recovered from the whole body, or it can be recovered from the eyebrows after forming the eyebrows.
  • the posterior silk gland is an elongated part at the rearmost part, which becomes about 20 cm when extended.
  • the fibroin protein which is the center of the eyebrows, is synthesized later.
  • the central silk gland is a thick part that is bent in an S shape in the central part, and when extended, it becomes about 6 cm.
  • the fibroin protein sent from the posterior silk gland is concentrated and stored, and shaped into fibers. It also secretes another silk protein, sericin. When spitting out eyebrows, it acts as an adhesive that holds fibroin proteins together.
  • the anterior silk gland is a thin tube that connects to the spout, which is about 4 cm in length, and becomes thinner toward the end. Molecules of liquid fibroin protein are stretched and aligned in a certain direction and aggregate with each other to further remove water. At the tip of the tube, it merges with another pair of tubes and is spit out from the spout to become a single eyebrows.
  • Silk moth stops eating mulberry at the end of the 5th instar (mature silk moth).
  • the body of the tussah is filled with a pair of organs (silk glands) that store a liquid (liquid silk) like starch syrup, which is the raw material for eyebrows.
  • the silk gland is connected to the spout at the mouth of the silk moth through a thin spit tube.
  • Liquid silk is stretched and hardened by passing through a thin spit tube to become eyebrows.
  • the eyebrows are pulled out from the silk glands one after another by a series of movements in which the thread spit out from the spit tube by the larva is attached to a nearby object, the head and chest are moved in a figure eight shape, and the thread is pulled. It is.
  • the term "silk moth-type sugar chain” refers to a sugar chain structure peculiar to glycoproteins produced in silk moth, and is typically trimannosyl core (itself), oligomannose-type sugar chain, and complex-type sugar chain. , Or its hybrid type.
  • the silk moth-type glycoprotein is produced using the central silk gland, unless otherwise specified, the "silk moth-type sugar chain” refers to the specific sugar chain type produced by the central silk gland. Say.
  • Examples of such a silkworm-type sugar chain include two N-acetylglucosamines (GlcNAc) bound to asparagine (Asn) and then three molecular bindings of mannose (Man) (referred to as trimannosyl core, described below. It has a structure branched from the core (represented by Eq. (1)), and various sugar chains are further bound to it.
  • GlcNAc N-acetylglucosamines
  • Man mannose
  • a "corresponding" amino acid or nucleic acid has, or has, in a polypeptide molecule or a polynucleotide molecule, the same action as a given amino acid or nucleotide in a polypeptide or polynucleotide that serves as a reference for comparison.
  • the corresponding amino acids are specified to be, for example, cysteineized, glutathioneized, SS bond formed, oxidized (eg, methionine side chain oxidation), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc.
  • the corresponding amino acid can be the amino acid responsible for dimerization.
  • Such "corresponding" amino acids or nucleic acids may be regions or domains over a range. Thus, such cases are referred to herein as "corresponding" regions or domains.
  • a "corresponding" gene eg, a polypeptide molecule or a polynucleotide molecule
  • a gene to be produced for example, a polypeptide molecule or a polynucleotide molecule
  • the gene corresponding to a gene can be the ortholog of that gene.
  • mouse and rat RAGEs or soluble forms of sRAGE
  • corresponding genes can be identified using techniques well known in the art.
  • the corresponding gene in an animal eg, mouse
  • the reference gene for the corresponding gene eg, RAGE or soluble form of sRAGE
  • uses the sequence of the animal as a query sequence For example, it can be found by searching the sequence database of humans and rats).
  • biological function refers to a specific function that a gene, nucleic acid molecule or polypeptide can have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto. Examples include, but are not limited to, specific antibody production, enzymatic activity, and resistance imparting. In the present disclosure, for example, the function of RAGE to recognize a marker such as hemopexin can be mentioned, but the present invention is not limited thereto. As used herein, biological function can be exerted by "biological activity”.
  • biological activity refers to the activity that a certain factor (for example, polynucleotide, protein, etc.) can have in vivo, and exerts various functions (for example, transcription promoting activity). Activities include, for example, the activity of activating or inactivating another molecule by interacting with one molecule. When two factors interact, their biological activity is the binding between the two molecules and the resulting biological changes, eg, when one molecule is precipitated with an antibody to the other. When the molecules also coprecipitate, the two molecules are considered to be bound. Therefore, seeing such coprecipitation is one of the judgment methods. For example, if a factor is an enzyme, its biological activity comprises that enzymatic activity. In another example, when a factor is a ligand, the ligand involves binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.
  • activity indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus).
  • Various measurable indicators such as the affinity of a compound that binds directly to a polypeptide or polynucleotide of the present disclosure, or, for example, the amount of upstream or downstream protein or other after some stimulation or event. A measure of similar function can be mentioned.
  • the term "subject” refers to an organism (for example, a human) that is the subject of the diagnosis or detection of the present disclosure.
  • sample refers to any substance obtained from a subject or the like, and includes, for example, body fluids (blood, saliva, urine, tears, cerebrospinal fluid, etc.).
  • drug As used herein, “drug”, “drug” or “factor” (both of which correspond to agents in English) are used interchangeably as long as they can achieve their intended purpose. It may also be a substance or other element (eg, energy such as light, radioactivity, heat, electricity). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, cDNA, DNA such as genomic DNA, RNA such as mRNA), poly.
  • cDNA DNA such as genomic DNA
  • RNA such as mRNA
  • oligosaccharides lipids, organic small molecules (eg, hormones, ligands, signaling substances, organic small molecules, molecules synthesized with combinatorial chemistries, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.))
  • these complex molecules include, but are not limited to.
  • Factors specific to a polynucleotide typically include polynucleotides that have certain sequence homology (eg, 70% or more sequence identity) and complementarity to the sequence of the polynucleotide. Examples include, but are not limited to, polypeptides such as transcription factors that bind to the promoter region.
  • Factors specific for a polypeptide are typically an antibody or derivative thereof or an analog thereof (eg, a single chain antibody) specifically directed to that polypeptide, which is accepted by the polypeptide.
  • Specific ligands or receptors in the case of a body or ligand, substrates thereof when the polypeptide is an enzyme, and the like are included, but are not limited thereto.
  • interaction refers to two substances, that is, a force (for example, an intermolecular force (Van der Waals force), a hydrogen bond, a hydrophobic interaction) between one substance and the other substance. Etc.). Usually, the two interacting substances are in an associated or bound state.
  • binding means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions and the like. Physical interactions (bindings) can be direct or indirect, the indirect being through or due to the effects of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and is not accompanied by other substantial chemical intermediates.
  • contacting means physics of a compound, either directly or indirectly, with respect to a polypeptide or polynucleotide capable of functioning as a marker, ligand, etc. of the present disclosure. It means to bring them closer to each other.
  • the polypeptide or polynucleotide can be present in many buffers, salts, solutions and the like. Contact includes placing the compound in, for example, a beaker, a microtiter plate, a cell culture flask or a microarray (eg, a gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.
  • the present disclosure is an oxidized LDL or stimulating AGEs useful for diagnosing diseases (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease) and assessing the effectiveness of treatment. Is used to detect.
  • liver disease refers to any disease in the liver.
  • the liver disease targeted by the present invention can be any liver disease, but the liver disease can be a chronic fatty liver disease or an acute fatty liver disease, which can be an inflammatory disease or is related to lifestyle. It can be an inflammatory disease, a non-alcoholic disease, or a non-viral disease.
  • the present invention may be useful in diagnosing "non-alcoholic steatohepatitis (NAFL) and non-alcoholic steatohepatitis (NASH)" as well as diseases such as liver cirrhosis and hepatocellular carcinoma in which NASH is further advanced. ..
  • chronic fatty liver disease refers to a condition in which a large amount of fat is chronically accumulated in the liver. Chronic means that symptoms develop gradually and treatment and course are long-term. Factors that cause chronic diseases include, but are not limited to, age, gender, lifestyle, genetic factors, obesity, various hormonal abnormalities, and intake of some drugs. Therefore, chronic fatty liver disease is a disease that is completely different from "acute" fatty liver disease, has different pathological conditions and causes, and has different treatment and prevention methods.
  • inflammatory disease refers to a disease that accompanies inflammation
  • inflammatory disease refers to any fatty liver disease that accompanies inflammation. It is understood that fatty liver disease, which is an inflammatory disease, also includes non-alcoholic steatohepatitis (NASH) and the corresponding alcoholic steatohepatitis.
  • NASH non-alcoholic steatohepatitis
  • the term "lifestyle-related inflammatory disease” refers to a disease that is accompanied by inflammation, with lifestyle as the main factor. Lifestyle-related habits in the present invention include, but are not limited to, exercise amount, nutritional balance, smoking, alcohol intake, sleep time, and the like. "Inflammatory disease” is used in the sense commonly used in the art and means a disease characterized by a local response of tissue to the injury of a living body. In certain embodiments, the inflammatory disease in the present invention is a liver-related inflammatory disease.
  • non-alcoholic disease is a general term for diseases whose main factor is alcohol intake. Subjects with non-alcoholic diseases include not only subjects who do not consume alcohol at all, but also subjects who consume a small amount of alcohol (in men, the weight of pure ethanol consumed per day is less than 30 g). Yes, in women, the weight of pure ethanol ingested per day is less than 20 g). Non-alcoholic diseases are also collectively referred to as non-alcoholic steatohepatitis (NAFLD), and typical non-alcoholic diseases include non-alcoholic steatohepatitis (NAFL) and non-alcoholic steatohepatitis (NASH). Be done.
  • NAFLD non-alcoholic steatohepatitis
  • NASH non-alcoholic steatohepatitis
  • non-alcoholic fatty liver As used herein, the term "non-alcoholic fatty liver (NAFL)" is used in the sense commonly used in the art and is characterized by non-alcoholic and accumulation of fat in the liver. However, it means a disease in which inflammatory cells do not infiltrate the liver. NAFL is a disease with relatively mild symptoms and a good prognosis among fatty liver diseases, but it may progress to more severe diseases such as NASH or cirrhosis. In contrast to non-alcoholic fatty liver, there is also a disease called "alcoholic fatty liver” whose main factor is alcohol intake. Non-alcoholic fatty liver and alcoholic fatty liver differ in the presence or absence of alcohol intake, but the pathophysiology of these fatty livers is similar, and these differences may disappear 20 years after illness. It has been reported.
  • non-alcoholic steatosis is used in the sense commonly used in the art, including fat accumulation in hepatocytes, hepatocyte balloon-like degeneration (baluning degeneration), and apoptosis.
  • NASH non-alcoholic steatosis
  • hCLS fatty degenerated hepatocytes
  • the term "Mattenoni classification” is used in the sense commonly used in the art and is a classification method for determining the severity of fatty liver disease based on the condition of fatty liver disease.
  • the Mattenoni classification includes (1) hepatocyte steatosis, (2) inflammatory cell infiltration, (3) hepatocyte balloon-like swelling, and (4) liver fibers. It is a method of classifying NAFLD into four types, Type 1 to Type 4, based on the presence or absence of fibrosis and (5) Mallory-Denk form.
  • Matteni Type 1 shows only hepatocyte fat degeneration
  • Type 2 shows only hepatocyte fat degeneration with infiltration of inflammatory cells
  • Type 3 shows hepatocyte balloon-like swelling
  • Type 4 is added to Type 3.
  • Hepatocytes 3 and 4 are diagnosed as NASH.
  • liver fibrosis refers to the formation of excess fibrous connective tissue during the repair or reaction process of the liver. Fibrosis itself does not cause symptoms, but when fibrosis progresses severely, it causes liver cirrhosis and complications, and as a result, symptoms develop. There are many types of fibrosis and many causes. For example, in NASH, fibrosis is seen when it progresses to type 4. At this stage, the risk of developing cirrhosis and liver cancer must also be considered. Fibrosis is also caused by chemical substances (for example, carbon tetrachloride) (which can be expressed as acute fibrosis), but no fat accumulation in the liver is observed, and the chronic fatty liver of the present invention is not observed.
  • chemical substances for example, carbon tetrachloride
  • fibrosis in disease which can be described as chronic fibrosis.
  • acute fibrosis and chronic fibrosis are completely different pathological conditions in terms of their prevention and treatment because their pathological aspects are completely different.
  • the fibrotic tissue that replaced the hepatocytes does not have the function of hepatocytes.
  • fibrotic tissue impedes blood flow to and in the liver, limiting the supply of blood to hepatocytes and leading to hepatocyte death, thereby leading to further development of fibrosis. obtain.
  • the present invention can also predictively diagnose such fibrosis.
  • diagnosis refers to identifying various parameters related to a disease, disorder, condition, etc. in a subject and determining the current state or future of such a disease, disorder, condition.
  • condition within the body can be investigated and such information can be used to formulate a disease, disorder, condition, treatment to be administered or prevention in the subject.
  • various parameters such as a method can be selected.
  • diagnosis means diagnosing the current state, but in a broad sense, it includes “predictive diagnosis", "pre-diagnosis” and the like. Early diagnosis is sometimes called “early diagnosis”.
  • pre-diagnosis refers to liver disease, diabetes, diabetes using molecules that are interchangeably used and capable of recognizing LDL or AGEs (CTLD14, sRAGE, etc.).
  • CLD14 LDL or AGEs
  • sRAGE LDL or AGEs
  • Detecting the stage determining the risk of developing the disease in the future, and suffering from diabetes for the purpose of preventing diabetic complications such as liver disease, diabetes, diabetic nephropathy, diabetic nephropathy, and diabetic neuropathy. Includes determining if there is a risk of
  • the condition in the body can be investigated in advance, and such information can be used to determine the disease, disorder, condition in the subject.
  • Various parameters can be selected, such as the procedure or method for treatment or prevention to be administered.
  • "predictive diagnosis” or “pre-diagnosis” is used in part with the concept of "early diagnosis” because it also includes diagnosis at a stage that cannot be diagnosed by other conventional methods.
  • the diagnostic method of the present disclosure is industrially useful because it can be used from the body and can be carried out without the hands of medical professionals such as doctors.
  • "predictive diagnosis, pre-diagnosis or diagnosis” may be referred to as "support” in order to clarify that it can be carried out without the hands of medical personnel such as doctors.
  • "diagnosis” also includes assessing the effectiveness of treatment.
  • the term "detector” broadly refers to any factor capable of detecting a substance of interest (eg, oxidized LDL or irritant AGEs associated with a disease).
  • diagnosis agent is broadly defined as a condition of interest (eg, disease (eg, dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease, etc.)). Any factor that can diagnose.
  • measurement is used in the usual sense used in the art, and means to measure and obtain the amount of a certain object.
  • detection is used in the usual meaning used in the art, and means to inspect and find a substance, a component, etc.
  • identity is an existing substance related to a certain object.
  • identity is an existing substance related to a certain object.
  • it means determining the identity of the target substance as a chemical substance (for example, determining the chemical structure).
  • Quantitative means determining the amount of a substance of interest present.
  • detection or quantification by formation of a conjugate with (molecule) means that the detection or quantification of an object to be detected or quantified forms a conjugate with an entity different from the object. It means that whether or not it is used as an index. There may be a case where conjugate formation is used as an index and a case where inhibition of conjugate formation (using a competitive molecule) is used as an index.
  • treatment refers to a disease or disorder that, when such a condition occurs, prevents the exacerbation of the disease or disorder, preferably maintains the status quo, more preferably alleviates, and further. Preferably, it means to withdraw.
  • prevention refers to a disease (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease) or disorder before it becomes such a condition. It means not to become.
  • a disease such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease
  • it is possible to prevent diseases or disorders related to oxidized LDL or irritating AGEs, or to take preventive measures.
  • diabetic complications such as diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy, or to take measures for prevention. ..
  • the present disclosure is a device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule, including a membrane that develops a sample by capillary phenomenon.
  • the membrane specifically binds to a detection unit containing a detection binder that specifically binds to a biomarker molecule or a competing molecule thereof, and a binding molecule having an abnormal form of the biomarker molecule or a binding molecule capable of forming a conjugate with the competing molecule thereof.
  • the kit or device includes a control part containing a control binder to be bound, and the kit or device includes a sample contact part and fluorescent nanoparticles as a part or a separate element of a membrane.
  • the membrane may include a detection unit and a control unit in this order from upstream to downstream.
  • the sample contact, detection, and control units may be arranged or connected to each other so that the sample penetrates into each other by capillarity.
  • the biomarker molecules can be LDL or AGEs, and the aberrant forms of the biomarker molecules can be denatured LDL or stimulating AGEs.
  • the binding molecule can be CTLD14 or sRAGE.
  • the detection binding agent can be an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, and the detection target is irritating.
  • the detection target is AGEs, it can be an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof.
  • kits or devices of the present disclosure may or may not further include competing molecules of biomarker molecules.
  • the biomarker molecule is LDL
  • the variant of the biomarker molecule is denatured LDL
  • the binding molecule is CTLD14
  • the detection binding agent is an anti-LDL antibody, anti-denatured LDL antibody or anti-ApoB antibody. Or they can be antigen-binding fragments.
  • the biomarker molecule is AGEs
  • the variant of the biomarker molecule is stimulating AGEs
  • the binding molecule is sRAGE
  • the detection binding agent is an anti-BSA antibody or anti-OVA antibody.
  • the kit or device further comprises a competing molecule of the biomarker molecule, which competing molecule can be G-BSA or G-OVA.
  • the fluorescent nanoparticles can be provided as a detection reagent.
  • the detection reagents may be provided separately from the devices or kits of the present disclosure, or may be provided together with the devices or kits of the present disclosure.
  • the detection reagent may be mixed with the sample and used, or may be included in the conjugate portion. When the detection reagent is mixed with the sample, the conjugate section may be omitted.
  • the membrane When the sample is developed in the horizontal direction, the membrane preferably has a conjugate part containing the detection reagent, and when the sample is developed in the vertical direction, the conjugate part is omitted and the detection reagent is contained in the sample. It is preferable to mix them in advance.
  • composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising fluorescent nanoparticles, said device, system or kit.
  • a membrane that develops a sample by capillary phenomenon, and the membrane contains a detection binder that specifically binds to a biomarker molecule or a competing molecule thereof, and an abnormal form of the biomarker molecule or its competition.
  • a composition comprising a control moiety containing a control binder that specifically binds to a molecule and a binding molecule capable of forming a conjugate, wherein the fluorescent nanoparticles are used by labeling the binding molecule.
  • the device, system or kit may include the sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
  • the disclosure is a composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising a binding molecule labeled with fluorescent nanoparticles.
  • the binding molecule has the ability to form a conjugate with an atypical form of the biomarker molecule or a competing molecule thereof
  • the device, system or kit comprises a membrane that develops the sample by capillary phenomenon, and the membrane is the biomarker molecule.
  • a composition comprising a detection unit containing a detection binder that specifically binds to the competing molecule and a control unit containing a control binder that specifically binds to the binding molecule is provided.
  • the device, system or kit may include the sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
  • the present disclosure is a system or device for detecting or quantifying modified LDL that comprises a membrane that develops a sample by capillary phenomenon, wherein the membrane comprises CTLD14 labeled with fluorescent nanoparticles.
  • a system including a conjugate part, a detection part containing an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, and a control part containing a binding molecule to CTLD14.
  • the system or device may include sample contacts and fluorescent nanoparticles as part of or as a separate element of the membrane. The system of the present disclosure can detect denatured LDL easily and quickly as compared with the conventional method.
  • the system of the present disclosure can be a lateral flow assay type system.
  • the system of the present disclosure is advantageous because it is possible to detect denatured LDL in a contaminating blood sample without the need for additional steps to remove the contaminants.
  • the conjugate portion may be provided as a separate element from the membrane.
  • the sample contact portion of the present disclosure is a portion to which a sample (for example, blood, etc.) can be contacted, and may have any shape as long as the sample comes into contact with the sample, and the material may be any material. There may be, but materials that react with the sample can be avoided if possible.
  • the sample contact portion may be provided as a part of the membrane or as a separate element, but in any case, the sample contact portion is connected to the control portion and the detection portion so as to penetrate the sample by capillarity. There is a need.
  • the sample contact may include a blood cell separation filter.
  • the blood cell separation filter refers to a filter for filtering red blood cells, white blood cells, and platelets and sending out components other than blood cells to the membrane, and examples thereof include, but are not limited to, FUSION5, LF1, MF1, and VF2. ..
  • the blood cell separation filter can be FUSION 5 or LF1.
  • the conjugate portion in the present disclosure includes a fluorescent nanoparticle-labeled CTLD14.
  • the conjugate portion may be composed of a structure and a material in which the fluorescent nanoparticle labeled CTLD14 and the sample can be contacted.
  • the fluorescent nanoparticles can be selected from compound semiconductor nanoparticles or fluorescent latex particles, preferably amorphous silica particles, most preferably silanol groups with a highly hydrophilic particle surface.
  • Fluorescent silica nanoparticles for example, Quartz Dot
  • the fluorescent nanoparticle labeled CTLD14 is arranged in the conjugate section.
  • CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strept-tagged, in which case the binding molecule to CTLD14 is streptavidin. , Anti-His antibody, anti-Myc antibody, anti-Flag antibody, anti-E-tag antibody, or Strept-Tactin.
  • CTLD14 may have silk moth-type sugar chains.
  • CTLD14 having such a silk moth type sugar chain may be produced by silk moth or may be artificially added with a sugar chain, but it is preferably produced by silk moth.
  • CTLD14 produced by silk moths co-expressing biotin ligase can be biotinylated by including a biotinylated tag within the sequence of CTLD14.
  • the tag sequence to be biotinylated is BioEase. tag, Avi. Examples include, but are not limited to, tag, any sequence capable of undergoing biotinylation.
  • biotinylated CTLD14 By using biotinylated CTLD14, it is possible to use streptavidin, which is relatively easily available, in the control section.
  • the obtained biotinylated CTLD14 is stable and excellent even when conjugated with fluorescent nanoparticles under alkaline conditions.
  • CTLD14 has at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, about 100% identity with the amino acid sequence of SEQ ID NO: 2. obtain.
  • a method for producing biotinylated CTLD14 having a silk moth-type sugar chain is also described in detail in WO 2016/051808, which is incorporated herein by reference.
  • the detector (test spot or line) comprises an anti-denatured LDL antibody, an anti-LDL antibody or an anti-ApoB antibody.
  • the detection unit is a part that enables detection of whether or not a target component (for example, denatured LDL, stimulating AGEs) is present in a sample via these antibodies. Therefore, any shape and material that enables detection may be used.
  • control section is the part that confirms that the sample has been developed on the device and that the binding molecule to CTLD14 (eg, antibody to CTLD14, or biotinylated CTLD14). If so, it contains streptavidin) that detects this biotin.
  • the detection unit test spot or line
  • the molecule binding to CTLD14 binds to CTLD14 and then or in parallel with the antibody, so that the fluorescent nanoparticle-labeled CTLD14 aggregates to increase the fluorescence intensity and the target component. Allows detection or quantification. Therefore, it may be made of any shape and material that enables binding, control and detection. Detection or quantification is also described in detail in (Detection or Quantification Method).
  • the present disclosure is a system for detecting or quantifying stimulating AGEs, which comprises a membrane that develops a sample by capillarity, wherein the membrane comprises a fluorescent nanoparticle labeled sRAGE.
  • a system comprising a detection unit containing an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and a control unit containing a binding molecule to sRAGE.
  • the conjugate portion may further comprise a G-BSA or G-OVA.
  • the conjugate portion may be provided as a separate element from the membrane.
  • stimulating AGEs in a sample can be detected by reducing the spot density (fluorescence intensity) on the test line due to competition for sRAGE.
  • spot density fluorescence intensity
  • stimulating AGEs in a sample can be detected by reducing the spot density (fluorescence intensity) on the test line due to competition for sRAGE.
  • the fluorescent nanoparticle-labeled sRAGE binds to G-BSA and fluoresces because it is captured and aggregated by the antibody on the test line, whereas in the presence of stimulating AGEs in the sample, G-BSA It is intended that the amount of binding to sRAGE is reduced, and as a result, aggregation of fluorescent nanoparticle-labeled sRAGE on the test line is less likely to occur, and as a result, the fluorescence of the test spot is reduced.
  • the presence of stimulating AGEs can be detected by the degree of inhibition of the fluorescence intensity of this test line.
  • casein, PVA, or the like can be appropriately used because BSA cannot be used in the conjugate buffer or blocking buffer.
  • a system in which an anti-OVA (ovalbumin) antibody is used for the test line and G-OVA is used as competitive AGEs can also be implemented.
  • the system or device of the present disclosure may preferably include a blood cell separator.
  • the blood cell separator is desirable when the sample contains or is expected to contain blood cells or blood components. It may be particularly advantageous to be able to separate blood cells, as blood may have components that inhibit detection.
  • the present disclosure is a device or kit for detecting or quantifying anomalous forms of a biomarker molecule by conjugate formation with the biomarker molecule, including a membrane that develops a sample by capillary phenomenon.
  • a detection unit containing a competing molecule of a biomarker molecule, and a control unit containing a control binder that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having the ability to form a conjugate with the competing molecule.
  • the kit or device comprises a sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
  • FIG. 17 shows a schematic diagram of a detection system containing a competing molecule in the detection unit.
  • the biomarker molecule is AGEs
  • the variant of the biomarker molecule is stimulating AGEs
  • the binding molecule is sRAGE
  • the competing molecules are BSA without glycation, OVA without glycation, CML.
  • - Can be BSA, CML-OVA, G-BSA or G-OVA.
  • the competing molecule can be unsaccharified BSA, unsaccharified OVA, CML-BSA or CML-OVA, preferably unsaccharified BSA or CML-BSA, most preferably saccharified. It can be untreated BSA or CML-BSA.
  • the present disclosure is a method for detecting or quantifying anomalies of a biomarker molecule, in which the step of providing a sample is mixed with the bound molecule labeled with fluorescent nanoparticles.
  • the binding molecule has the ability to form a conjugate with an atypical form of the biomarker molecule or a competing molecule thereof, and the step and the sample contact portion of the membrane in the device or kit of the present disclosure are mixed.
  • a method including a step of contacting a sample and a step of adding a buffer solution as needed after the contact.
  • the present disclosure is a method for detecting or quantifying denatured LDL or irritating AGEs, the step of providing a blood sample and the blood sample with a buffer and an anticoagulant (eg, heparin).
  • a method comprising a step of mixing the blood sample, a step of bringing the sample contact portion in the system or device of the present disclosure into contact with the mixed blood sample, and a step of adding the buffer solution after the contact. ..
  • the buffer solution includes, but is not limited to, phosphate buffered saline (PBS) and the like.
  • the buffer can be PBS ( ⁇ ).
  • the buffer may or may not contain albumin such as bovine serum albumin (BSA), casein, and other proteins (which do not affect subsequent manipulation) such as PVA.
  • the buffer can be BSA-added phosphate buffered saline (PBS) ( ⁇ ).
  • Example 1 Purification of a single-chain antibody from a silk moth central silk gland extract
  • GAL4 biotin ligase
  • biotinylated single chain antibody by feeding 5th instar larvae with a diet supplemented with 20 ⁇ g of biotin per 1 g and supplying biotin necessary for biotinization of the anti-LDL single chain antibody. ..
  • a non-biotinylated single chain antibody was used.
  • the central silk gland was removed and the protein was extracted with Triton X-100 / PBS (-).
  • TALON (Clontech) resin equilibrated with PBS metal chelate affinity chromatography resin using Co
  • PBS metal chelate affinity chromatography resin using Co
  • imidazole was adjusted to a final concentration of 5 mM.
  • the resin was washed with PBS containing 10 mM imidazole and then the single chain antibody was eluted with PBS containing 1 M imidazole.
  • the eluted fraction was collected and dialyzed against PBS (-).
  • a sample after dialysis was concentrated using a centrifugal ultrafiltration filter (Amicon Ultra, Merc), and the buffer was further replaced with 10 mM phosphate buffer (pH 7.5).
  • CTLD14 which is a ligand recognition site of the LOX-1 receptor that recognizes oxidized LDL
  • silica nanoparticles (QuartzDot, Furukawa AE). The preparation method was carried out according to the instruction manual of QuartzDot, and the outline is as follows.
  • QuartzDot (100 nm particle size) is mixed with dimethylformamide containing N- (4-aminophenyl) maleimide (final concentration 2 mg / ml) in a tube to a final concentration of 2 mg / ml and incubated at 30 ° C. for 30 minutes. , Centrifuge and remove the supernatant.
  • MES buffer pH 6.0, final concentration 100 mM
  • N-hydroxysuccinimide final concentration 23 mg / ml
  • hydrochloric acid-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide final concentration 2.88 mg
  • CTLD14 solution final concentration 30 ⁇ g / ml
  • Blocking agents A and B and MilliQ water were added to the precipitate at a ratio of 1: 2: 7, ultrasonically dispersed, and then centrifuged to remove the supernatant.
  • the precipitate was ultrasonically dispersed with 10 mM phosphate buffer (pH 7.5), centrifuged to remove the supernatant, and the phosphate buffer was added again and ultrasonically dispersed to obtain a fluorescent nanoparticle-labeled CTLD14.
  • Streptavidin as a control spot and 0.5 ⁇ l of a single-chain antibody as a test spot were each spotted on a half-strip membrane consisting of an absorption pad and a membrane, and dried at 37 ° C. for 1 h. After the spot was dried, it was immersed in a 1% BSA / PBS solution at room temperature and gently shaken for 15 minutes, lightly washed twice with MilliQ water, and then gently shaken for another 5 minutes for washing. After washing, it was lightly drained and air-dried overnight on a paper towel with a Kimwipe. A fluorescence detector is required for detection, but a simple detector is sufficient. In this example, a fluorescence image analyzer: Typhoon was used.
  • FIG. 2 shows that oxidized LDL can be detected using fluorescent silica nanoparticle labeled CTLD14.
  • FIG. 3 shows the results of measuring the fluorescence intensity of each spot. It has been clarified that it is possible to quantitatively indicate the oxidized LDL concentration based on the fluorescence intensity.
  • Modification with fluorescent silica nanoparticles must be performed in salt-free buffer (PB, not PBS). Since the silk moth MSG-derived CTLD14 was able to maintain its function even in the presence of non-salt, it could be modified with fluorescent silica nanoparticles, and it was possible to develop a lateral flow assay system that can be quantified by measuring the fluorescence intensity.
  • PB salt-free buffer
  • Example 3 Detection of added oxidized LDL by immunochromatometry
  • a single chain antibody was applied to the test spot, and streptavidin was applied to the control spot.
  • Oxidized LDL was added to 75 ⁇ l of PBS (containing 0.5% BSA) to prepare a model sample.
  • 2 ⁇ l of fluorescent nanoparticle labeled CTLD14 was added to the model sample in a microtube and mixed, it was transferred into the well of a 96-well microplate, stripped and vertically developed. The development was completed after about 30 minutes, and the fluorescence intensity was measured at the same time as the fluorescence image was acquired by TyphoonFLA9600.
  • Example 4 Comparison of single chain antibody spots by immunochromatographic assay using serum of hyperlipidemic patients
  • the values of LDL, HDL, and TG are abnormal. It was classified into levels 0 to 4 according to whether it was in the value, boundary area, or management target value. Furthermore, the correlation between each classification and the oxidized LDL value (measured according to International Publication No. 2016/051808) was analyzed. Next, the oxidized LDL in each level of serum was measured according to Example 2.
  • FIG. 7 shows the fluorescence image after development, and the right figure shows the fluorescence intensity.
  • the sample group (serum 2 to 6) having an item classified as abnormal with respect to level 0 "serum 1"
  • the fluorescence intensity of the spot was strong. Therefore, it was suggested that the control state can be known from the simple and rapid quantification value of the oxidized LDL concentration.
  • Oxidized LDL and fluorescent nanoparticle-labeled CTLD14 are added to a microtube containing whole blood, mixed, transferred to a well of a microplate, and a full strip with a blood cell removal filter (a single chain antibody having a different concentration in a test spot). was applied to the control spot with streptavidin) and developed in the vertical direction.
  • a blood cell removal filter a single chain antibody having a different concentration in a test spot
  • Example 6 Preparation of fluorescent nanoparticle labeled sRAGE for AGE detection using silica nanoparticles
  • the method for preparing the fluorescent nanoparticles labeled sRAGE using silica nanoparticles was carried out by modifying the instruction manual of QuartzDot. The outline is as follows.
  • QuartzDot (100 nm particle size) is mixed with dimethylformamide containing N- (4-aminophenyl) maleimide (final concentration 2 mg / ml) in a tube to a final concentration of 2 mg / ml and incubated at 30 ° C. for 30 minutes. , Centrifuge and remove the supernatant.
  • MES buffer pH 6.0, final concentration 100 mM
  • N-hydroxysuccinimide final concentration 23 mg / ml
  • hydrochloric acid-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide final concentration 2.88 mg
  • the mixture was incubated at 30 ° C. for 1 hour, centrifuged, and the supernatant was removed.
  • the blocking agent is a mixture of blocking agents A, B and MilliQ water attached to QuartzDot in a ratio of 1: 2: 7, or a phosphate buffer containing 0.05-1.0% PVA. Tris buffer, or phosphate buffer or Tris buffer containing 0.05-2.5% casein was used. A blocking agent was added, and the mixture was ultrasonically dispersed and then centrifuged to remove the supernatant. The precipitate was ultrasonically dispersed with 10 mM phosphate buffer (pH 7.5), centrifuged to remove the supernatant, and the phosphate buffer was added again and ultrasonically dispersed to obtain a fluorescent nanoparticle-labeled sRAGE.
  • Fluorescent nanoparticle-labeled sRAGE is added to a microtube containing whole blood, mixed, transferred to a well of a microplate, and a full strip with a blood cell removal filter (no antibody application to the test spot, streptavidin in the control spot). Was inserted) and developed in the vertical direction.
  • Example 7 Detection of AGEs by lateral flow detection method using fluorescent nanoparticles
  • Preparation of AGE Detection Reagent (Silica Nanoparticle Labeled sRAGE) Using Fluorescent Nanoparticles Fluorescent nanoparticles (QuartzDot, Furukawa Denko Advanced Engineering) Labeled sRAGE Preparation method was performed by modifying the instruction manual of QuartzDot. The outline is as follows.
  • QuartzDot (100 nm particle size) is mixed with dimethylformamide containing N- (4-aminophenyl) maleimide (final concentration 2 mg / ml) in a tube to a final concentration of 2 mg / ml and incubated at 30 ° C. for 30 minutes. , Centrifuge and remove the supernatant.
  • MES buffer pH 6.0, final concentration 100 mM
  • N-hydroxysuccinimide final concentration 23 mg / ml
  • hydrochloric acid-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide final concentration 2.88 mg
  • sRAGE solution final concentration 30-50 ⁇ g / ml
  • the blocking agent was a mixture of blocking agents A and B attached to QuartzDot and MilliQ water in a ratio of 1: 2: 7, or a phosphate buffer containing 0.1-0.5% PVA.
  • a phosphate buffer containing 0.5-1.0% casein was used.
  • a blocking agent was added, and the mixture was ultrasonically dispersed and then centrifuged to remove the supernatant.
  • the precipitate was ultrasonically dispersed with 10 mM phosphate buffer (pH 7.5), centrifuged to remove the supernatant, and the phosphate buffer was added again and ultrasonically dispersed to obtain a fluorescent nanoparticle-labeled sRAGE.
  • the cryopreserved serum was thawed in ice and centrifuged at 1,000 ⁇ g for 15 minutes, and 60 ⁇ l of the supernatant diluted 500 or 1000 times with PBS was used.
  • the detection reagent (QuartzDot-labeled sRAGE) was added to the sample and mixed, the mixed solution was transferred to a microplate well, and then the prepared half-strip for lateral flow was inserted. After 20 to 30 minutes had passed and all the mixed solution in the well was absorbed by the strip, the fluorescence intensity of the test spot and the control spot was confirmed using Typhoon FLA 9500.
  • an AGE solution was added to 75 ⁇ l of blood diluted with a 500-fold reaction buffer and mixed with a detection reagent (QuartzDot-labeled sRAGE).
  • the mixed solution was transferred to a microplate well, and a full strip with a blood cell separation pad was inserted into the sample pad portion.
  • After 5 to 10 minutes, when the strip has absorbed the mixture add 50 ⁇ l of reaction buffer to the wells, and after about 30 minutes, after all the solutions have been absorbed, test spots and controls using Typhoon FLA 9500. The fluorescence intensity of the spot was confirmed.
  • Model sample, AGE / PBS (-) Anti-AGEs antibody was applied to the test spot, and a sample (BSA without saccharification treatment or BSA with glucose saccharification) was added to the buffer solution for development. It was confirmed that the AGE bound to the fluorescent nanoparticle-labeled biotinylated sRAGE was bound to the test line and the fluorescence intensity was increased (image acquisition with an image analyzer) (FIG. 11A). Further, CML saccharification-BSA (CML-BSA, antagonistic AGEs) was applied to the test spot, and a sample (BSA without saccharification treatment or CML-BSA) was added to the buffer solution for development.
  • CML saccharification-BSA CML saccharification-BSA, antagonistic AGEs
  • FIG. 17 shows an outline of the detection system in which CML-BSA is applied to the test spot.
  • Detection of AGEs in Serum Anti-AGEs antibody was applied to the test spot, and a sample (BSA without glycation treatment or BSA with glucose glycation) was added to human serum for development. It was confirmed that the AGE bound to the fluorescent nanoparticle-labeled biotinylated sRAGE was bound to the test line and the fluorescence intensity was increased (Fig. 12A). Further, CML-BSA (antagonistic AGEs) was applied to the test spot, and a sample (BSA without saccharification treatment or CML BSA) was added to the buffer solution for development. Binding of fluorescent nanoparticle-labeled biotinylated sRAGE to the test line was inhibited by AGEs, and attenuation of fluorescence intensity depending on the concentration of AGEs was confirmed (Fig. 12B).
  • AGEs in whole blood Anti-AGEs antibody was applied to the test spot, and a sample (BSA without glycation treatment or BSA with glucose glycation) was added to fresh human blood for development. It was confirmed that the AGE bound to the fluorescent nanoparticle-labeled biotinylated sRAGE was bound to the test line and the fluorescence intensity was increased (FIG. 13A). Further, CML-BSA (antagonistic AGEs) was applied to the test spot, and a sample (BSA without saccharification treatment or CMLBSA) was added to fresh human blood for development. The binding of fluorescent nanoparticle-labeled biotinylated sRAGE to the test line was inhibited by AGEs, and a decrease in fluorescence intensity was confirmed (Fig. 13B).
  • CML-BSA antagonistic AGEs
  • NAFLD non-alcoholic fatty liver diseases
  • NASH nonalcoholic fatty liver diseases
  • This disclosure is useful in the field of disease diagnosis or predictive diagnosis.
  • SEQ ID NO: 1 Nucleic acid sequence encoding CTLD14
  • SEQ ID NO: 2 Amino acid sequence encoding CTLD14
  • SEQ ID NO: 3 RAGE nucleic acid sequence
  • SEQ ID NO: 4 RAGE amino nucleic acid sequence
  • SEQ ID NO: 5 sRAGE nucleic acid used in the present invention
  • SEQ ID NO: 6 Amino acid sequence of sRAGE used in the present invention
  • SEQ ID NO: 7 Amino acid sequence of the single-stranded antibody used in the present invention

Abstract

The present disclosure provides a technique for quantitatively detecting denatured LDL or stimulative AGEs in a quick and easy manner. The present disclosure also provides a device or kit for detecting or quantizing aberrant forms of biomarker molecules by forming a conjugate with the biomarker molecules, the device or kit including a membrane that carries out development of a specimen due to a capillary phenomenon, wherein: the membrane includes a detection part including a detection binding agent that specifically binds to the biomarker molecules or to competing molecules thereof, and a control part including a control binding agent that specifically binds to bound molecules and has a function for forming a conjugate with aberrant forms of the biomarker molecules or with competing molecules thereof; and the device or kit includes a specimen contact part and fluorescent nanoparticles as part of the membrane or as separate elements.

Description

変性ＬＤＬ及び刺激性ＡＧＥｓ簡易迅速定量法Simple and rapid quantification of denatured LDL and stimulant AGEs
　変性ＬＤＬまたは刺激性ＡＧＥｓを簡便迅速に定量検出する技術に関する。 The present invention relates to a technique for simply and quickly quantitatively detecting denatured LDL or stimulating AGEs.
　酸化ＬＤＬ（いわゆる「悪玉コレステロール」）は、脂質異常症、虚血性心疾患などの危険因子として知られているが、酸化ＬＤＬは、酸化的修飾を受けたＬＤＬ分子の総称であり、修飾の程度も異なった不均一な分子集団である。発明者らは、酸化的修飾を受けたＬＤＬ分子中から、生体中で疾病の引き金となる酸化ＬＤＬ（真の悪玉）のみを広範に検出する手法を開発に成功した（特許文献１）。しかし、開発された手法は、専門的な技術と装置が必要であり研究機関・検査機関等での使用を前提としている。一方、運動、食生活改善などの生活習慣の改善による疾病の進行抑制効果、投薬などによる疾病の管理状況を簡便迅速に定量評価する手法の開発が求められている。 Oxidized LDL (so-called "bad cholesterol") is known as a risk factor for dyslipidemia, ischemic heart disease, etc. Oxidized LDL is a general term for oxidatively modified LDL molecules and the degree of modification. Is also a different heterogeneous population of molecules. The inventors have succeeded in developing a method for widely detecting only oxidized LDL (true bad) that triggers a disease in a living body from oxidatively modified LDL molecules (Patent Document 1). However, the developed method requires specialized technology and equipment, and is premised on use in research institutes and inspection institutions. On the other hand, there is a need for the development of a method for easily and quickly quantitatively evaluating the effect of suppressing the progression of diseases by improving lifestyles such as exercise and dietary habits, and the management status of diseases by medication.
　ＡＧＥｓ（Ａｄｖａｎｃｅｄ　ｇｌｙｃａｔｉｏｎ　ｅｎｄ　ｐｒｏｄｕｃｔｓ：終末糖化産物）は、タンパク質の糖化産物であるが、多様な構造体の総称である。近年、ヒト体内にも糖化ストレスにより生じるＡＧＥｓが存在し、その中に糖尿病合併症、加齢性疾患（関節リュウマチ、アルツハイマー型認知症など）を誘発しうる刺激性ＡＧＥｓが含まれることが見いだされた。発明者らは、多様な構造のＡＧＥｓの中から、疾病の引き金となるＡＧＥｓを検出する手法の開発に成功した。しかし、開発された手法は、酸化ＬＤＬ検出法同様に、研究機関・検査機関等での使用を前提としており、運動、食生活改善などの生活習慣の改善による疾病の進行抑制効果、投薬などによる疾病の管理状況を簡便迅速に定量評価する手法の開発が求められている。 AGEs (advanced glycation end products: advanced glycation end products) are glycation products of proteins, but are a general term for various structures. In recent years, it has been found that AGEs caused by glycation stress also exist in the human body, and include stimulating AGEs that can induce diabetic complications and age-related diseases (rheumatoid arthritis, Alzheimer's disease, etc.). It was. The inventors have succeeded in developing a method for detecting AGEs that trigger diseases from among AGEs having various structures. However, the developed method is premised on use in research institutes, inspection institutions, etc., like the oxidized LDL detection method, and is based on the effect of suppressing the progression of diseases by improving lifestyle such as exercise and dietary habits, and by medication. There is a need to develop a method for quantitative and quick quantitative evaluation of disease management status.
国際公開第２０１６／０５１８０８号International Publication No. 2016/051808
　本発明者らは、酸化ＬＤＬまたは刺激性ＡＧＥｓを定量することができる技術、例えばラテラルフローアッセイ（イムノクロマト法）を開発した。具体的には、当該定量技術において、酸化ＬＤＬまたは刺激性ＡＧＥｓを認識する蛍光ナノ粒子で修飾された認識素子を使用することによって、簡便迅速な酸化ＬＤＬまたは刺激性ＡＧＥｓの定量化が達成された。 The present inventors have developed a technique capable of quantifying oxidized LDL or stimulating AGEs, for example, a lateral flow assay (immunochromatography). Specifically, in the quantification technique, simple and rapid quantification of oxidized LDL or stimulating AGEs was achieved by using a recognition element modified with fluorescent nanoparticles that recognize oxidized LDL or stimulating AGEs. ..
　したがって、本開示は、例えば、以下を提供する。
（項目１）
　毛管現象による試料の展開を行うメンブレンを含む、生体マーカー分子の異常型を該生体マーカー分子とのコンジュゲート形成により検出または定量するためのデバイスまたはキットであって、該メンブレンが、
　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含み、
　該キットまたはデバイスは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含む、キットまたはデバイス。
（項目２）
　前記メンブレンが、上流から下流にかけて、前記検出部、および前記コントロール部をこの順番で含む、項目１に記載のキットまたはデバイス。
（項目３）
　前記試料接触部、前記検出部、および前記コントロール部が、相互に毛管現象で試料が浸透するように配置または連結されている、項目１および１Ａに記載のキットまたはデバイス。
（項目４）
前記生体マーカー分子はＬＤＬまたはＡＧＥｓである、項目１～３のいずれか一項に記載のデバイスまたはキット。
（項目５）
前記生体マーカー分子の異常型は変性ＬＤＬまたは刺激性ＡＧＥｓである、項目１～４のいずれか一項に記載のデバイスまたはキット。
（項目６）
前記結合分子はＣＴＬＤ１４またはｓＲＡＧＥである、項目１～５のいずれか一項に記載のデバイスまたはキット。
（項目７）
前記検出用結合剤は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメント、あるいは、抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントである、項目１～６のいずれか一項に記載のデバイスまたはキット。
（項目８）
前記キットまたはデバイスは、前記生体マーカー分子の競合分子をさらに含む、項目１～７のいずれか一項に記載のデバイスまたはキット。
（項目９）
前記生体マーカー分子はＬＤＬであり、前記生体マーカー分子の異常型は変性ＬＤＬであり、前記結合分子はＣＴＬＤ１４であり、前記検出用結合剤は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメントである、項目１～８のいずれか一項に記載のデバイスまたはキット。
（項目１０）
前記生体マーカー分子はＡＧＥｓであり、前記生体マーカー分子の異常型は刺激性ＡＧＥｓであり、前記結合分子はｓＲＡＧＥであり、前記検出用結合剤は抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントであり、前記キットまたはデバイスは、前記生体マーカー分子の競合分子をさらに含み、該競合分子はＧ－ＢＳＡもしくはＧ－ＯＶＡである、項目１～８のいずれか一項に記載のデバイスまたはキット。
（項目１１）
前記蛍光ナノ粒子は、検出試薬として提供される、項目１～１０のいずれか一項に記載のデバイスまたはキット。
（項目１２）
前記蛍光ナノ粒子は、前記メンブレン中で、試料混合物として提供される、項目１～１０のいずれか一項に記載のデバイスまたはキット。
（項目１３）
前記試料接触部が、血球分離部を含む、項目１～１２のいずれか一項に記載のデバイスまたはキット。
（項目１４）
前記血球分離部が、ＦＵＳＩＯＮ５、ＬＦ１、ＭＦ１、およびＶＦ２から選択される、項目１３に記載のデバイスまたはキット。
（項目１５）
前記ＣＴＬＤ１４が、ビオチン化、Ｈｉｓタグ付加、Ｍｙｃタグ付加、Ｆｌａｇタグ付加、Ｅタグ付加、またはＳｔｒｅｐタグ付加なされており、それぞれの場合において、前記コントロール用結合剤は、ストレプトアビジン、抗Ｈｉｓ抗体、抗Ｍｙｃ抗体、抗Ｆｌａｇ抗体、抗Ｅタグ抗体、またはＳｔｒｅｐ－Ｔａｃｔｉｎである、項目４に記載のデバイスまたはキット。
（項目１６）
前記ＣＴＬＤ１４が、カイコ型糖鎖を有する、項目６に記載のデバイスまたはキット。
（項目１７）
前記ＣＴＬＤ１４がビオチン化されており、前記コントロール用結合剤が、ストレプトアビジンである、項目６に記載のデバイスまたはキット。
（項目１８）
前記試料が、血液試料である、項目１～１７のいずれか一項に記載のデバイスまたはキット。
（項目１９）
前記血液試料が、血清または全血である、項目１８に記載のデバイスまたはキット。
（項目２０）
生体マーカー分子の異常型を検出または定量するための方法であって、
　試料を提供する工程と、
　該試料と蛍光ナノ粒子で標識された結合分子とを混合する工程であって、該結合分子は生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する、工程と、
　項目１～１８のいずれか一項に記載のデバイスまたはキットにおける前記メンブレンの前記試料接触部と、該混合された試料とを接触させる工程と、
　接触後、必要に応じて緩衝液を添加する工程と
を含む、方法。
（項目２１）
　蛍光ナノ粒子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、
　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
　試料接触部と
　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含み、
　該蛍光ナノ粒子は、該結合分子を標識することにより使用される、
組成物。
（項目２２）
　蛍光ナノ粒子で標識された結合分子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、該結合分子は、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有し、
　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
　試料接触部と
　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
　該結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含む、
組成物。
（項目２３）
　毛管現象による試料の展開を行うメンブレンを含む、生体マーカー分子の異常型を該生体マーカー分子とのコンジュゲート形成により検出または定量するためのデバイスまたはキットであって、該メンブレンが、
　生体マーカー分子の競合分子を含む検出部と
　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含み、
　該キットまたはデバイスは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含む、キットまたはデバイス。
（項目２４）
　上記１または複数の項目の特徴を有する、請求項２３に記載のキットまたはデバイス。
（項目２５）
　蛍光ナノ粒子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、
　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
　試料接触部と
　生体マーカー分子の競合分子含む検出部と
　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含み、
　該蛍光ナノ粒子は、該結合分子を標識することにより使用される、
組成物。
（項目２６）
　蛍光ナノ粒子で標識された結合分子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、該結合分子は、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有し、
　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
　試料接触部と
　生体マーカー分子の競合分子を含む検出部と
　該結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含む、
組成物。
（項目２７）
　上記１または複数の項目の特徴を有する、請求項２５または２６に記載の組成物。
（項目２８）
　生体マーカー分子の異常型を検出または定量するための方法であって、
　試料を提供する工程と、
　該試料と蛍光ナノ粒子で標識された結合分子とを混合する工程であって、該結合分子は生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する、工程と、
　項目２３に記載のデバイスまたはキットにおける前記メンブレンの前記試料接触部と、該混合された試料とを接触させる工程と、
　接触後、必要に応じて緩衝液を添加する工程と
を含む、方法。
（項目２９）
　上記１または複数の項目の特徴を有する、請求項２８に記載の方法。 Thus, the present disclosure provides, for example,:
(Item 1)
A device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule, which comprises a membrane for developing a sample by capillarity, wherein the membrane is:
A detection unit containing a detection binder that specifically binds to a biomarker molecule or a competing molecule thereof, and a control bond that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having the ability to form a conjugate with the competing molecule. Including the control part containing the agent,
The kit or device comprises a sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
(Item 2)
The kit or device according to item 1, wherein the membrane includes the detection unit and the control unit in this order from upstream to downstream.
(Item 3)
The kit or device according to items 1 and 1A, wherein the sample contact portion, the detection portion, and the control portion are arranged or connected to each other so that the sample permeates through a capillary phenomenon.
(Item 4)
The device or kit according to any one of items 1 to 3, wherein the biological marker molecule is LDL or AGEs.
(Item 5)
The device or kit according to any one of items 1 to 4, wherein the aberrant form of the biomarker molecule is denatured LDL or stimulating AGEs.
(Item 6)
The device or kit according to any one of items 1 to 5, wherein the binding molecule is CTLD14 or sRAGE.
(Item 7)
The detection binding agent is any one of items 1 to 6, which is an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, or an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof. The device or kit described in the section.
(Item 8)
The device or kit according to any one of items 1 to 7, wherein the kit or device further comprises a competing molecule of the biomarker molecule.
(Item 9)
The biomarker molecule is LDL, the variant of the biomarker molecule is denatured LDL, the binding molecule is CTLD14, and the detection binding agent is anti-LDL antibody, anti-modified LDL antibody or anti-ApoB antibody or them. The device or kit according to any one of items 1 to 8, which is an antigen-binding fragment of.
(Item 10)
The biomarker molecule is AGEs, the variant of the biomarker molecule is stimulating AGEs, the binding molecule is sRAGE, and the detection binding agent is an anti-BSA antibody or anti-OVA antibody or an antigen-binding fragment thereof. The device or kit according to any one of items 1 to 8, wherein the kit or device further comprises a competing molecule of the biomarker molecule, wherein the competing molecule is G-BSA or G-OVA.
(Item 11)
The device or kit according to any one of items 1 to 10, wherein the fluorescent nanoparticles are provided as a detection reagent.
(Item 12)
The device or kit according to any one of items 1 to 10, wherein the fluorescent nanoparticles are provided as a sample mixture in the membrane.
(Item 13)
The device or kit according to any one of items 1 to 12, wherein the sample contact portion includes a blood cell separation portion.
(Item 14)
13. The device or kit of item 13, wherein the blood cell separator is selected from FUSION5, LF1, MF1, and VF2.
(Item 15)
The CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case, the control binder is streptavidin, an anti-His antibody, The device or kit of item 4, which is an anti-Myc antibody, an anti-Flag antibody, an anti-E-tag antibody, or a Strept-Tactin.
(Item 16)
The device or kit according to item 6, wherein the CTLD 14 has a silk moth-type sugar chain.
(Item 17)
The device or kit of item 6, wherein the CTLD14 is biotinylated and the control binder is streptavidin.
(Item 18)
The device or kit according to any one of items 1 to 17, wherein the sample is a blood sample.
(Item 19)
The device or kit of item 18, wherein the blood sample is serum or whole blood.
(Item 20)
A method for detecting or quantifying abnormal types of biological marker molecules.
The process of providing the sample and
A step of mixing the sample with a binding molecule labeled with fluorescent nanoparticles, wherein the binding molecule has the ability to form a conjugate with an aberrant form of a biomarker molecule or a competing molecule thereof.
A step of contacting the sample contact portion of the membrane with the mixed sample in the device or kit according to any one of items 1 to 18.
A method comprising the step of adding a buffer as needed after contact.
(Item 21)
A composition for use in a device, system or kit for detecting or quantifying anomalies of biomarker molecules, including fluorescent nanoparticles.
The device, system or kit comprises a membrane that develops a sample by capillary action.
The sample contact part and the detection part containing a detection binder that specifically binds to the biomarker molecule or its competing molecule and the abnormal type of the biomarker molecule or the binding molecule having the ability to form a conjugate with the competing molecule Includes a control unit containing a control binder
The fluorescent nanoparticles are used by labeling the binding molecule.
Composition.
(Item 22)
A composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising a binding molecule labeled with fluorescent nanoparticles, said binding molecule of the biomarker molecule. Has the ability to form conjugates with atypical or competing molecules thereof,
The device, system or kit comprises a membrane that develops a sample by capillary action.
It includes a sample contact part, a detection part containing a detection binder that specifically binds to a biological marker molecule or a competing molecule thereof, and a control part that contains a control binder that specifically binds to the binding molecule.
Composition.
(Item 23)
A device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule, which comprises a membrane for developing a sample by capillarity, wherein the membrane is:
It includes a detection part containing a competing molecule of a biomarker molecule and a control part containing a control binder that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having a conjugate forming ability with the competing molecule.
The kit or device comprises a sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
(Item 24)
23. The kit or device according to claim 23, which has the characteristics of one or more of the above items.
(Item 25)
A composition for use in a device, system or kit for detecting or quantifying anomalies of biomarker molecules, including fluorescent nanoparticles.
The device, system or kit comprises a membrane that develops a sample by capillary action.
Includes a sample contact part, a detection part containing a competing molecule of a biomarker molecule, and a control part containing a control binder that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having the ability to form a conjugate with the competing molecule. ,
The fluorescent nanoparticles are used by labeling the binding molecule.
Composition.
(Item 26)
A composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising a binding molecule labeled with fluorescent nanoparticles, said binding molecule of the biomarker molecule. Has the ability to form conjugates with atypical or competing molecules thereof,
The device, system or kit comprises a membrane that develops a sample by capillary action.
It includes a sample contact part, a detection part containing a competing molecule of a biological marker molecule, and a control part containing a control binder that specifically binds to the binding molecule.
Composition.
(Item 27)
The composition according to claim 25 or 26, which has the characteristics of the above one or more items.
(Item 28)
A method for detecting or quantifying abnormal types of biological marker molecules.
The process of providing the sample and
A step of mixing the sample with a binding molecule labeled with fluorescent nanoparticles, wherein the binding molecule has the ability to form a conjugate with an aberrant form of a biomarker molecule or a competing molecule thereof.
The step of contacting the sample contact portion of the membrane with the mixed sample in the device or kit according to item 23.
A method comprising the step of adding a buffer as needed after contact.
(Item 29)
28. The method of claim 28, which has the characteristics of one or more of the above items.
　さらに、本発明は、以下の項目を提供する。
（項目１Ａ）
　毛管現象による試料の展開を行うメンブレンを含む、生体マーカー分子の異常型を該生体マーカー分子とのコンジュゲート形成により検出または定量するためのデバイスまたはキットであって、該メンブレンが、
　試料接触部と
　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含み、
　該キットまたはデバイスは、蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含む、キットまたはデバイス。
（項目２Ａ）
前記生体マーカー分子はＬＤＬまたはＡＧＥｓである、項目１Ａに記載のデバイスまたはキット。
（項目３Ａ）
前記生体マーカー分子の異常型は変性ＬＤＬまたは刺激性ＡＧＥｓである、項目１Ａに記載のデバイスまたはキット。
（項目４Ａ）
前記結合分子はＣＴＬＤ１４またはｓＲＡＧＥである、項目１Ａ～３Ａのいずれか一項に記載のデバイスまたはキット。
（項目５Ａ）
前記検出用結合剤は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメント、あるいは、抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントである、項目１Ａ～４Ａのいずれか一項に記載のデバイスまたはキット。
（項目６Ａ）
前記キットまたはデバイスは、前記生体マーカー分子の競合分子をさらに含む、項目１Ａ～５Ａのいずれか一項に記載のデバイスまたはキット。
（項目７Ａ）
前記生体マーカー分子はＬＤＬであり、前記生体マーカー分子の異常型は変性ＬＤＬであり、前記結合分子はＣＴＬＤ１４であり、前記検出用結合剤は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメントである、項目１Ａ～６Ａのいずれか一項に記載のデバイスまたはキット。
（項目８Ａ）
前記生体マーカー分子はＡＧＥｓであり、前記生体マーカー分子の異常型は刺激性ＡＧＥｓであり、前記結合分子はｓＲＡＧＥであり、前記検出用結合剤は抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントであり、前記キットまたはデバイスは、前記生体マーカー分子の競合分子をさらに含み、該競合分子はＧ－ＢＳＡもしくはＧ－ＯＶＡである、項目１Ａ～６Ａのいずれか一項に記載のデバイスまたはキット。
（項目９Ａ）
前記蛍光ナノ粒子は、検出試薬として提供される、項目１Ａ～８Ａのいずれか一項に記載のデバイスまたはキット。
（項目１０Ａ）
前記蛍光ナノ粒子は、前記メンブレン中で、試料混合物として提供される、項目１Ａ～８Ａのいずれか一項に記載のデバイスまたはキット。
（項目１１Ａ）
前記メンブレンが、血球分離部をさらに含む、項目１Ａ～１０Ａのいずれか一項に記載のデバイスまたはキット。
（項目１２Ａ）
前記血球分離部が、ＦＵＳＩＯＮ５、ＬＦ１、ＭＦ１、およびＶＦ２から選択される、項目１１Ａに記載のデバイスまたはキット。
（項目１３Ａ）
前記ＣＴＬＤ１４が、ビオチン化、Ｈｉｓタグ付加、Ｍｙｃタグ付加、Ｆｌａｇタグ付加、Ｅタグ付加、またはＳｔｒｅｐタグ付加なされており、それぞれの場合において、前記コントロール用結合剤は、ストレプトアビジン、抗Ｈｉｓ抗体、抗Ｍｙｃ抗体、抗Ｆｌａｇ抗体、抗Ｅタグ抗体、またはＳｔｒｅｐ－Ｔａｃｔｉｎである、項目４Ａに記載のデバイスまたはキット。
（項目１４Ａ）
前記ＣＴＬＤ１４が、カイコ型糖鎖を有する、項目４Ａに記載のデバイスまたはキット。
（項目１５Ａ）
前記ＣＴＬＤ１４がビオチン化されており、前記コントロール用結合剤が、ストレプトアビジンである、項目４Ａに記載のデバイスまたはキット。
（項目１６Ａ）
前記試料が、血液試料である、項目１Ａ～１５Ａのいずれか一項に記載のデバイスまたはキット。
（項目１７Ａ）
生体マーカー分子の異常型を検出または定量するための方法であって、
　試料を提供する工程と、
　該試料と蛍光ナノ粒子で標識された結合分子とを混合する工程であって、該結合分子は生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する、工程と、
　項目１Ａ～１６Ａのいずれか一項に記載のデバイスまたはキットにおける前記メンブレンの前記試料接触部と、該混合された試料とを接触させる工程と、
　接触後、必要に応じて緩衝液を添加する工程と
を含む、方法。
（項目１８Ａ）
　蛍光ナノ粒子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、
　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
　試料接触部と
　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含み、
　該蛍光ナノ粒子は、該結合分子を標識することにより使用される、
組成物。
（項目１９Ａ）
　蛍光ナノ粒子で標識された結合分子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、該結合分子は、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有し、
　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
　試料接触部と
　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
　該結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
を含む、組成物。 Furthermore, the present invention provides the following items.
(Item 1A)
A device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule, which comprises a membrane for developing a sample by capillarity, wherein the membrane is:
The sample contact part and the detection part containing a detection binder that specifically binds to the biomarker molecule or its competing molecule and the abnormal type of the biomarker molecule or the binding molecule having the ability to form a conjugate with the competing molecule Includes a control unit containing a control binder
The kit or device comprises fluorescent nanoparticles as part of or as a separate element of the membrane.
(Item 2A)
The device or kit of item 1A, wherein the biomarker molecule is LDL or AGEs.
(Item 3A)
The device or kit of item 1A, wherein the aberrant form of the biomarker molecule is denatured LDL or stimulating AGEs.
(Item 4A)
The device or kit according to any one of items 1A to 3A, wherein the binding molecule is CTLD14 or sRAGE.
(Item 5A)
The detection binding agent is any one of items 1A to 4A, which is an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, or an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof. The device or kit described in the section.
(Item 6A)
The device or kit according to any one of items 1A to 5A, wherein the kit or device further comprises a competing molecule of the biomarker molecule.
(Item 7A)
The biomarker molecule is LDL, the variant of the biomarker molecule is denatured LDL, the binding molecule is CTLD14, and the detection binding agent is anti-LDL antibody, anti-modified LDL antibody or anti-ApoB antibody or them. The device or kit according to any one of items 1A to 6A, which is an antigen-binding fragment of.
(Item 8A)
The biomarker molecule is AGEs, the variant of the biomarker molecule is stimulating AGEs, the binding molecule is sRAGE, and the detection binding agent is an anti-BSA antibody or anti-OVA antibody or an antigen-binding fragment thereof. The device or kit according to any one of items 1A to 6A, wherein the kit or device further comprises a competing molecule of the biomarker molecule, wherein the competing molecule is G-BSA or G-OVA.
(Item 9A)
The device or kit according to any one of items 1A to 8A, wherein the fluorescent nanoparticles are provided as a detection reagent.
(Item 10A)
The device or kit according to any one of items 1A to 8A, wherein the fluorescent nanoparticles are provided as a sample mixture in the membrane.
(Item 11A)
The device or kit according to any one of items 1A to 10A, wherein the membrane further comprises a blood cell separator.
(Item 12A)
The device or kit of item 11A, wherein the blood cell separator is selected from FUSION5, LF1, MF1, and VF2.
(Item 13A)
The CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case, the control binder is streptavidin, an anti-His antibody, The device or kit of item 4A, which is an anti-Myc antibody, an anti-Flag antibody, an anti-E-tag antibody, or Strept-Tactin.
(Item 14A)
The device or kit of item 4A, wherein the CTLD 14 has a silk moth-type sugar chain.
(Item 15A)
The device or kit of item 4A, wherein the CTLD14 is biotinylated and the control binder is streptavidin.
(Item 16A)
The device or kit according to any one of items 1A to 15A, wherein the sample is a blood sample.
(Item 17A)
A method for detecting or quantifying abnormal types of biological marker molecules.
The process of providing the sample and
A step of mixing the sample with a binding molecule labeled with fluorescent nanoparticles, wherein the binding molecule has the ability to form a conjugate with an aberrant form of a biomarker molecule or a competing molecule thereof.
A step of contacting the sample contact portion of the membrane with the mixed sample in the device or kit according to any one of items 1A to 16A.
A method comprising the step of adding a buffer as needed after contact.
(Item 18A)
A composition for use in a device, system or kit for detecting or quantifying anomalies of biomarker molecules, including fluorescent nanoparticles.
The device, system or kit comprises a membrane that develops a sample by capillary action.
The sample contact part and the detection part containing a detection binder that specifically binds to the biomarker molecule or its competing molecule and the abnormal type of the biomarker molecule or the binding molecule having the ability to form a conjugate with the competing molecule Includes a control unit containing a control binder
The fluorescent nanoparticles are used by labeling the binding molecule.
Composition.
(Item 19A)
A composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising a binding molecule labeled with fluorescent nanoparticles, said binding molecule of the biomarker molecule. Has the ability to form conjugates with atypical or competing molecules thereof,
The device, system or kit comprises a membrane that develops a sample by capillary action.
A composition comprising a sample contact portion, a detection portion containing a detection binder that specifically binds to a biological marker molecule or a competing molecule thereof, and a control portion that contains a control binder that specifically binds to the binding molecule.
　本開示において、上記１または複数の特徴は、明示された組み合わせに加え、さらに組み合わせて提供され得ることが意図される。本開示のなおさらなる実施形態および利点は、必要に応じて以下の詳細な説明を読んで理解すれば、当業者に認識される。 In the present disclosure, it is intended that the above one or more features may be provided in a further combination in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.
　本開示によれば、疾患に関連する酸化ＬＤＬまたは刺激性ＡＧＥｓを簡便かつ迅速に定量することができる技術が提供される。したがって、本開示は、疾患（脂質異常症、糖尿病合併症、肝疾患、および、アルツハイマー型認知症など）を診断、治療の有効性を評価、予防措置を講ずるのに有用な酸化ＬＤＬまたは刺激性ＡＧＥｓを検出するためのキットおよび方法、ならびにそれに使用することができる基板を提供する。 According to the present disclosure, there is provided a technique capable of easily and quickly quantifying oxidized LDL or stimulant AGEs associated with a disease. Therefore, the present disclosure is an oxidized LDL or irritant useful for diagnosing diseases (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer-type dementia), assessing the effectiveness of treatment, and taking preventive measures. Kits and methods for detecting AGEs, as well as substrates that can be used for it are provided.
図１は、一本鎖抗体産生の概要を示す。FIG. 1 shows an outline of single chain antibody production. 図２は、ラテラルフロー（イムノクロマト）アッセイによる酸化ＬＤＬの検出結果を示す。FIG. 2 shows the results of detection of oxidized LDL by the lateral flow (immunochromatography) assay. 図３は、図２におけるストリップの各スポットの蛍光強度を測定した結果を示す。FIG. 3 shows the results of measuring the fluorescence intensity of each spot of the strip in FIG. 図４は、イムノクロマトアッセイによる血清中の添加酸化ＬＤＬ検出結果を示す。FIG. 4 shows the results of detection of added oxidized LDL in serum by immunochromatography assay. 図５は、ラテラルフローアッセイによる変性ＬＤＬの検出の原理の概略図を示す。FIG. 5 shows a schematic diagram of the principle of detection of denatured LDL by lateral flow assay. 図６は、高脂血症患者血清中の酸化ＬＤＬ濃度と脂質異常症患者のＬＤＬ，ＨＤＬ，および、ＴＧ濃度から判断される管理状態との相関を示す。脂質異常患者のＬＤＬ，ＨＤＬ，ＴＧの値が管理目標値以内であるか、異常であると判断される値であるかによる分類（左）に基づき、ＬＤＬ、ＨＤＬ、ＴＧの値の内、異常と分類される項目数などを参考に、患者血清をレベル０～４に分類し（右下段）、レベル毎の酸化ＬＤＬ濃度の範囲を整理した（右上段）。FIG. 6 shows the correlation between the oxidized LDL concentration in the serum of hyperlipidemic patients and the management status judged from the LDL, HDL, and TG concentrations of dyslipidemia patients. Among the LDL, HDL, and TG values, abnormalities are based on the classification (left) based on whether the LDL, HDL, and TG values of patients with dyslipidemia are within the control target values or are judged to be abnormal. Patient sera were classified into levels 0 to 4 (lower right), and the range of oxidized LDL concentration for each level was arranged (upper right) with reference to the number of items classified as. 図７は、図６の分類の高脂血症患者の血清を用いたイムノクロマトアッセイによる酸化ＬＤＬ濃度の比較を示す。FIG. 7 shows a comparison of oxidized LDL concentrations by an immunochromatographic assay using sera of hyperlipidemic patients classified in FIG. 図８は、イムノクロマトアッセイによる全血試料への添加酸化ＬＤＬの検出結果を示す。FIG. 8 shows the results of detection of oxidized LDL added to whole blood samples by immunochromatography assay. 図９は、蛍光ナノ粒子標識ｓＲＡＧＥが、イムノクロマト上で全血添加時に正常に展開可能か確認した結果である。FIG. 9 shows the results of confirming on immunochromatography whether the fluorescent nanoparticle-labeled sRAGE can be normally developed when whole blood is added. 図１０は、ラテラルフローアッセイによる刺激性ＡＧＥsの検出の原理の概略図を示す。FIG. 10 shows a schematic diagram of the principle of detection of stimulating AGEs by the lateral flow assay. 図１１は、モデルサンプルを用いたラテラルフローアッセイの結果を示す。FIG. 11 shows the results of a lateral flow assay using a model sample. 図１２は、ラテラルフローアッセイによる血清中のＡＧＥｓの検出結果を示す。図１２の左の画像は、イメージアナライザー取得した画像を示し、右のグラフは、各スポットの蛍光強度を示す。FIG. 12 shows the results of detection of AGEs in serum by the lateral flow assay. The image on the left of FIG. 12 shows the image acquired by the image analyzer, and the graph on the right shows the fluorescence intensity of each spot. 図１３は、ラテラルフローアッセイによる全血中のＡＧＥｓの検出結果を示す。図１３の左の画像は、イメージアナライザー取得した画像を示し、右のグラフは、各スポットの蛍光強度を示す。FIG. 13 shows the results of detection of AGEs in whole blood by the lateral flow assay. The image on the left of FIG. 13 shows an image acquired by an image analyzer, and the graph on the right shows the fluorescence intensity of each spot. 図１４は、ラテラルフローアッセイによる全血中のＡＧＥｓの検出結果を示す。図１４の左の画像は、イメージアナライザー取得した画像を示し、右のグラフは、各スポットの蛍光強度を示す。FIG. 14 shows the results of detection of AGEs in whole blood by the lateral flow assay. The image on the left of FIG. 14 shows the image acquired by the image analyzer, and the graph on the right shows the fluorescence intensity of each spot. 図１５は、ラテラルフローアッセイによるＮＡＳＨ患者血清中のＡＧＥｓの検出結果を示す。図１５の左の画像は、イメージアナライザー取得した画像を示し、右のグラフは、各スポットの蛍光強度を示す。FIG. 15 shows the results of detection of AGEs in the serum of NASH patients by the lateral flow assay. The image on the left of FIG. 15 shows the image acquired by the image analyzer, and the graph on the right shows the fluorescence intensity of each spot. 図１６は、ラテラルフローアッセイによる糖尿病合併症患者血清中のＡＧＥｓの検出結果を示す。図１６の左の画像は、イメージアナライザー取得した画像を示し、右のグラフは、各スポットの蛍光強度を示す。FIG. 16 shows the results of detection of AGEs in the serum of diabetic complication patients by the lateral flow assay. The image on the left of FIG. 16 shows an image acquired by an image analyzer, and the graph on the right shows the fluorescence intensity of each spot. 図１７は、検出部（テストスポット）に競合分子（ＣＭＬ糖化ＢＳＡ）が塗布された検出系の概要図を示す。FIG. 17 shows a schematic diagram of a detection system in which a competing molecule (CML saccharified BSA) is applied to a detection unit (test spot).
　以下、本開示を説明する。本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。従って、単数形の冠詞（例えば、英語の場合は「a」、「an」、「the」など）は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用されるすべての専門用語および科学技術用語は、本開示の属する分野の当業者によって一般的に理解されるのと同じ意味を有する。矛盾する場合、本明細書（定義を含めて）が優先する。 The present disclosure will be described below. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the plural concept unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.
　（用語の定義）
　以下に本明細書において特に使用される用語の定義を列挙する。 (Definition of terms)
The following is a list of definitions of terms specifically used herein.
　本明細書において、「約」とは、示される値の±１０％を意味する。 In the present specification, "about" means ± 10% of the indicated value.
　本明細書において、「システム」とは、検出、予測診断、事前診断、診断等を行うための任意の系をいい、一般に、１または複数の構成要素からなり、複数の構成要素がある場合それらの要素は互いに作用・関連し合っており、全体として調和のとれた挙動・機能を示すという３条件を満足する系をいう。システムは、装置、デバイス、組成物、診断薬など任意の形態であり得る。従って、システムは、例えば、測定装置を備える大掛かりなシステムから、クロマトグラフィーを備えるシステム、免疫反応を利用したキット・組み合わせ物、抗体を含む組成物（すなわち、マーカーのモノクローナル抗体を含む、体外医薬品である診断薬）などを包含することが理解される。 As used herein, the term "system" refers to any system for performing detection, predictive diagnosis, pre-diagnosis, diagnosis, etc., and generally consists of one or more components, and if there are a plurality of components, those. The elements of are acting and related to each other, and refer to a system that satisfies the three conditions of exhibiting harmonious behavior and function as a whole. The system can be in any form such as a device, device, composition, diagnostic agent. Thus, the system can be, for example, from a large-scale system with a measuring device, to a system with chromatography, a kit / combination utilizing an immune reaction, a composition containing an antibody (ie, an in vitro drug containing a monoclonal antibody of a marker). It is understood to include certain diagnostic agents) and the like.
　本明細書において「デバイス」とは、検出、予測診断、事前診断、診断等を行うための任意の装置をいい、１または複数の構成要素からなり、複数の構成要素がある場合通常それらの要素が互いに連絡されていることが多く、特定の目的で用いられる装置、器具、道具、物全般を指す意味で用いられ、機械的または電気的作用を有するものに限定解釈されるものではない。通常目的（例えば、検査、検出、診断など）を可能にするように互いに作動可能に連結されている少なくとも１つの要素を含む。 As used herein, the term "device" refers to any device for performing detection, predictive diagnosis, pre-diagnosis, diagnosis, etc., and is composed of one or a plurality of components, and if there are a plurality of components, those components are usually used. Are often in contact with each other and are used to refer to devices, appliances, tools, and objects used for a specific purpose in general, and are not construed as being limited to those having mechanical or electrical action. Includes at least one element that is operably linked to each other to enable normal purpose (eg, examination, detection, diagnosis, etc.).
　本明細書において「キット」とは、通常2つ以上の区画に分けて、提供されるべき部分
（例えば、メンブレン、デバイス、試薬など）が提供されるユニットをいう。試薬やデバイスが独立して複数提供される場合、このキットとして提供されることが好都合であり得る。そのようなキットは、好ましくは、提供される部分（例えば、メンブレンやデバイス）をどのように使用するか、あるいは、試薬をどのように使用するかを記載する指示書または説明書を備えていることが有利である。 As used herein, the term "kit" refers to a unit that is usually divided into two or more compartments and is provided with parts to be provided (eg, membranes, devices, reagents, etc.). If multiple reagents or devices are provided independently, it may be convenient to provide them as this kit. Such kits preferably include instructions or instructions describing how to use the provided parts (eg, membranes or devices) or how to use the reagents. Is advantageous.
　本明細書において「指示書」は、本開示を使用する方法を使用者に対する説明を記載したものである。この指示書は、本開示の使用方法を指示する文言が記載されている。この指示書は、必要な場合は、本開示が実施される国の監督官庁（例えば、日本であれば厚生労働省または農林水産省等、米国であれば食品医薬品局（FDA）、農務省（USDA）など）が規定した様式に従って作成され、その監督官庁により承認を受けた旨が明記される。指示書は、紙媒体で提供され得るが、それに限定されず、例えば、電子媒体（例えば、インターネットで提供されるホームページ、電子メール、ＳＮＳ、簡易メッセージなど）のような形態でも提供され得る。 In the present specification, the "instruction" describes the method for using the present disclosure to the user. This instruction contains language that directs how to use this disclosure. If necessary, this instruction may be provided by the regulatory agency of the country in which this disclosure is implemented (eg, Ministry of Health, Labor and Welfare or Ministry of Agriculture, Forestry and Fisheries in Japan, Food and Drug Administration (FDA), Department of Agriculture (USDA) in the United States. ) Etc.), and it is clearly stated that it has been approved by the regulatory agency. Instructions may be provided in paper media, but are not limited to, and may also be provided in the form of, for example, electronic media (eg, homepages, e-mails, SNS, simple messages, etc. provided on the Internet).
　本明細書において、「蛍光ナノ粒子」とは、目的生体物質を検出するのに十分な強度の蛍光を発することができるナノサイズの粒子である。蛍光ナノ粒子として、好ましくは量子ドット（半導体ナノ粒子）、蛍光物質集積ナノ粒子が使用される。 In the present specification, the "fluorescent nanoparticles" are nanoparticles of nano-sized particles capable of emitting fluorescence having sufficient intensity to detect a target biological substance. As the fluorescent nanoparticles, quantum dots (semiconductor nanoparticles) and fluorescent substance-integrated nanoparticles are preferably used.
　量子ドットとしては、ＩＩ－ＶＩ族化合物、ＩＩＩ－Ｖ族化合物またはＩＶ族元素を含有する半導体ナノ粒子が使用される。たとえば、ＣｄＳｅ、ＣｄＳ、ＣｄＴｅ、ＺｎＳｅ、ＺｎＳ、ＺｎＴｅ、ＩｎＰ、ＩｎＮ、ＩｎＡｓ、ＩｎＧａＰ、ＧａＰ、ＧａＡｓ、Ｓｉ、Ｇｅなどが挙げられる。蛍光物質集積ナノ粒子は、有機物または無機物でできた粒子を母体とし、複数の蛍光物質（たとえば、上記量子ドット、蛍光色素など）がその中に内包されているおよび／またはその表面に吸着している構造を有する、ナノサイズの粒子である。 As the quantum dots, semiconductor nanoparticles containing a group II-VI compound, a group III-V compound, or a group IV element are used. For example, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, Ge and the like can be mentioned. Fluorescent substance-accumulated nanoparticles are based on particles made of organic or inorganic substances, and a plurality of fluorescent substances (for example, the quantum dots, fluorescent dyes, etc.) are contained therein and / or adsorbed on the surface thereof. It is a nano-sized particle having a structure.
　蛍光物質集積ナノ粒子としては、母体と蛍光物質とが、互いに反対の電荷を有する置換基または部位を有し、静電的相互作用が働くものであることが好適である。蛍光物質集積ナノ粒子としては、量子ドット集積ナノ粒子、蛍光色素集積ナノ粒子などが使用される。 As the fluorescent substance-accumulated nanoparticles, it is preferable that the mother body and the fluorescent substance have substituents or sites having opposite charges and electrostatically interact with each other. As the fluorescent substance integrated nanoparticles, quantum dot integrated nanoparticles, fluorescent dye integrated nanoparticles and the like are used.
　母体のうち、有機物としては、メラミン樹脂、尿素樹脂、アニリン樹脂、グアナミン樹脂、フェノール樹脂、キシレン樹脂、フラン樹脂など、一般的に熱硬化性樹脂に分類される樹脂；スチレン樹脂、アクリル樹脂、アクリロニトリル樹脂、ＡＳ樹脂（アクリロニトリル－スチレン共重合体）、ＡＳＡ樹脂（アクリロニトリル－スチレン－アクリル酸メチル共重合体）など、一般的に熱可塑性樹脂に分類される樹脂；ポリ乳酸等のその他の樹脂；多糖を例示することができる。母体のうち、無機物としては、シリカ、ガラスなどを例示することができる。 Among the mother bodies, the organic substances are resins generally classified as thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin; styrene resin, acrylic resin, and acrylonitrile. Resins generally classified as thermoplastic resins such as resins, AS resins (acrylonitrile-styrene copolymers), ASA resins (acrylonitrile-styrene-methyl acrylate copolymers); other resins such as polylactic acid; polysaccharides Can be exemplified. Examples of the inorganic substance in the mother body include silica and glass.
　量子ドット集積ナノ粒子
　量子ドット集積ナノ粒子とは、上記量子ドットが、上記母体の中に内包されている、および／またはその表面に吸着している構造を有する。量子ドットが母体に内包されている場合、量子ドットは母体内部に分散されていればよく、母体自体と化学的に結合していてもよいし、していなくてもよい。 Quantum dot integrated nanoparticles The quantum dot integrated nanoparticles have a structure in which the quantum dots are contained in the parent body and / or adsorbed on the surface thereof. When the quantum dots are contained in the mother body, the quantum dots may or may not be chemically bonded to the mother body itself as long as they are dispersed inside the mother body.
　蛍光色素集積ナノ粒子
　蛍光色素集積ナノ粒子とは、蛍光色素が、上記母体の中に内包されている、および／またはその表面に吸着している構造を有する。蛍光色素としては、ローダミン系色素分子、スクアリリウム系色素分子、シアニン系色素分子、芳香環系色素分子、オキサジン系色素分子、カルボピロニン系色素分子、ピロメセン系色素分子などを例示することができる。蛍光色素としては、Ａｌｅｘａ　Ｆｌｕｏｒ（登録商標、インビトロジェン社製）系色素分子、ＢＯＤＩＰＹ（登録商標、インビトロジェン社製）系色素分子、Ｃｙ（登録商標、ＧＥヘルスケア社製）系色素分子、ＨｉＬｙｔｅ（登録商標、アナスペック社製）系色素分子、ＤｙＬｉｇｈｔ（登録商標、サーモサイエンティフィック社製）系色素分子、ＡＴＴＯ（登録商標、ＡＴＴＯ－ＴＥＣ社製）系色素分子、ＭＦＰ（登録商標、Ｍｏｂｉｔｅｃ社製）系色素分子、ＣＦ（登録商標、Ｂｉｏｔｉｕｍ社製）系色素分子、ＤＹ（登録商標、ＤＹＯＭＩＣＳ社製）系色素分子、ＣＡＬ（登録商標、ＢｉｏＳｅａｒｃｈ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）系色素分子などを用いることができる。 Fluorescent dye-accumulated nanoparticles The fluorescent dye-accumulated nanoparticles have a structure in which a fluorescent dye is contained in the mother body and / or is adsorbed on the surface thereof. Examples of the fluorescent dye include rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic ring-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, and pyromesene-based dye molecules. Examples of the fluorescent dye include Alexa Fluor (registered trademark, Invigen) dye molecule, BODIPY (registered trademark, Invigen) dye molecule, Cy (registered trademark, GE Healthcare) dye molecule, and HiLite (registered). Trademarks, Anaspec) dye molecules, DyLight (registered trademark, Thermoscientific) dye molecules, ATTO (registered trademark, ATTO-TEC) dye molecules, MFP (registered trademark, Mobitec) ) Dye molecule, CF (registered trademark, manufactured by Biotium) dye molecule, DY (registered trademark, manufactured by DYOMICS) dye molecule, CAL (registered trademark, manufactured by BioSearch Technologies) dye molecule and the like can be used. ..
　具体的には、５－カルボキシ－フルオレセイン、６－カルボキシ－フルオレセイン、５，６－ジカルボキシ－フルオレセイン、６－カルボキシ－２’，４，４’，５’，７，７’－ヘキサクロロフルオレセイン、６－カルボキシ－２’，４，７，７’－テトラクロロフルオレセイン、６－カルボキシ－４’，５’－ジクロロ－２’，７’－ジメトキシフルオレセイン、ナフトフルオレセイン、５－カルボキシ－ローダミン、６－カルボキシ－ローダミン、５，６－ジカルボキシ－ローダミン、ローダミン６Ｇ、テトラメチルローダミン、Ｘ－ローダミン、スルホローダミンＢ、スルホローダミン１０１、及びＡｌｅｘａ　Ｆｌｕｏｒ３５０、Ａｌｅｘａ　Ｆｌｕｏｒ　４０５、Ａｌｅｘａ　Ｆｌｕｏｒ　４３０、Ａｌｅｘａ　Ｆｌｕｏｒ　４８８、Ａｌｅｘａ　Ｆｌｕｏｒ　５００、Ａｌｅｘａ　Ｆｌｕｏｒ　５１４、Ａｌｅｘａ　Ｆｌｕｏｒ　５３２、Ａｌｅｘａ　Ｆｌｕｏｒ　５４６、Ａｌｅｘａ　Ｆｌｕｏｒ　５５５、Ａｌｅｘａ　Ｆｌｕｏｒ　５６８、Ａｌｅｘａ　Ｆｌｕｏｒ　５９４、Ａｌｅｘａ　Ｆｌｕｏｒ　６１０、Ａｌｅｘａ　Ｆｌｕｏｒ　６３３、Ａｌｅｘａ　Ｆｌｕｏｒ　６３５、Ａｌｅｘａ　Ｆｌｕｏｒ　６４７、Ａｌｅｘａ　Ｆｌｕｏｒ　６６０、Ａｌｅｘａ　Ｆｌｕｏｒ　６８０、Ａｌｅｘａ　Ｆｌｕｏｒ　７００、Ａｌｅｘａ　Ｆｌｕｏｒ　７５０、ＢＯＤＩＰＹ　ＦＬ、ＢＯＤＩＰＹ　ＴＭＲ、ＢＯＤＩＰＹ　４９３／５０３、ＢＯＤＩＰＹ　５３０／５５０、ＢＯＤＩＰＹ　５５８／５６８、ＢＯＤＩＰＹ　５６４／５７０、ＢＯＤＩＰＹ　５７６／５８９、ＢＯＤＩＰＹ　５８１／５９１、ＢＯＤＩＰＹ　６３０／６５０、ＢＯＤＩＰＹ　６５０／６６５（以上インビトロジェン社製）、メトキシクマリン、クマリン６、クマリン７、スルホクマリン６、スルホクマリン７、エオジン、ＮＢＤ、ピレン、Ｃｙ５、Ｃｙ５．５、Ｃｙ７、ＨｉＬｙｔｅ　Ｆｌｕｏｒ　４８８、ＨｉＬｙｔｅ　Ｆｌｕｏｒ　５５５、ＨｉＬｙｔｅ　Ｆｌｕｏｒ　５９４、ＨｉＬｙｔｅ　Ｆｌｕｏｒ　６４７、ＨｉＬｙｔｅ　Ｆｌｕｏｒ　６８０、ＨｉＬｙｔｅ　Ｆｌｕｏｒ　７５０（登録商標、アナスペック社製）、ＤｙＬｉｇｈｔ　３５０、ＤｙＬｉｇｈｔ　４０５、ＤｙＬｉｇｈｔ　４８８、ＤｙＬｉｇｈｔ　５５０、ＤｙＬｉｇｈｔ　５９４、ＤｙＬｉｇｈｔ　６３３、ＤｙＬｉｇｈｔ　６５０、ＤｙＬｉｇｈｔ　６８０、ＤｙＬｉｇｈｔ　７５５、ＤｙＬｉｇｈｔ　８００（登録商標、サーモサイエンティフィック社製）、ＡＴＴＯ　３９０、ＡＴＴＯ　４２５、ＡＴＴＯ　４６５、ＡＴＴＯ　４８８、ＡＴＴＯ　４９５、ＡＴＴＯ　５１４、ＡＴＴＯ　５２０、ＡＴＴＯ　５３２、ＡＴＴＯ　Ｒｈｏ６Ｇ、ＡＴＴＯ　５４２、ＡＴＴＯ　５５０、ＡＴＴＯ　５６５、ＡＴＴＯ　Ｒｈｏ３Ｂ、ＡＴＴＯ　Ｒｈｏ１１、ＡＴＴＯ　Ｒｈｏ１２、ＡＴＴＯ　Ｔｈｉｏ１２、ＡＴＴＯ　Ｒｈｏ１０１、ＡＴＴＯ　５９０、ＡＴＴＯ　５９４、ＡＴＴＯ　Ｒｈｏ１３、ＡＴＴＯ　６１０、ＡＴＴＯ　６２０、ＡＴＴＯ　Ｒｈｏ１４、ＡＴＴＯ　６３３、ＡＴＴＯ　６４７、ＡＴＴＯ　６４７Ｎ、ＡＴＴＯ　６５５、ＡＴＴＯ　Ｏｘａ１２、ＡＴＴＯ　６６５、ＡＴＴＯ　６８０、ＡＴＴＯ　７００、ＡＴＴＯ　７２５、ＡＴＴＯ　７４０（登録商標、ＡＴＴＯ－ＴＥＣ社製）、ＭＦＰ４８８、ＭＦＰ５５５、ＭＦＰ５９０、ＭＦＰ６３１（登録商標、Ｍｏｂｉｔｅｃ社製）、ＣＦ３５０、ＣＦ４０５Ｓ、ＣＦ４０５Ｍ、ＣＦ４８８Ａ、ＣＦ５１４、ＣＦ５３２、ＣＦ５４３、ＣＦ５５５、ＣＦ５６８、ＣＦ５９４、ＣＦ６２０Ｒ、ＣＦ６３３、ＣＦ６４０Ｒ、ＣＦ６４７、ＣＦ６６０Ｃ、ＣＦ６６０Ｒ、ＣＦ６８０、ＣＦ６８０Ｒ、ＣＦ７５０、ＣＦ７７０、ＣＦ７９０（登録商標、Ｂｉｏｔｉｕｍ社製）、ＤＹ－３５０、ＤＹ－４０５、ＤＹ－４１５、ＤＹ－４８０ＸＬ、ＤＹ－４８１ＸＬ、ＤＹ－４８５ＸＬ、ＤＹ－４９０、ＤＹ－４９５、ＤＹ－５０５、ＤＹ－５００ＸＬ、ＤＹ－５１０ＸＬ、ＤＹ－５２０ＸＬ、ＤＹ－５２１ＸＬ、ＤＹ－５３０、ＤＹ－５４７Ｐ１、ＤＹ－５４９Ｐ１、ＤＹ－５５０、ＤＹ－５５４、ＤＹ－５５５、ＤＹ－５５６、ＤＹ－５６０、ＤＹ－５９０、ＤＹ－５９１、ＤＹ－５９４、ＤＹ－６０５、ＤＹ－６１０、ＤＹ－６１５、ＤＹ－６３０、ＤＹ－６３１、ＤＹ－６３２、ＤＹ－６３３、ＤＹ－６３４、ＤＹ－６３５、ＤＹ－６３６、ＤＹ－６４７Ｐ１、ＤＹ－６４８Ｐ１、ＤＹ－６４９Ｐ１、ＤＹ－６５０、ＤＹ－６５４、ＤＹ－６５１、ＤＹ－６５２、ＤＹ－６７５、ＤＹ－６７６、ＤＹ－６７７、ＤＹ－６７８、ＤＹ－６７９Ｐ１、ＤＹ－６８０、ＤＹ－６８１、ＤＹ－６８２、ＤＹ－７００、ＤＹ－７０１、ＤＹ－７０３、ＤＹ－７０４、ＤＹ－７３０、ＤＹ－７３１、ＤＹ－７３２、ＤＹ－７３４、ＤＹ－７４９Ｐ１、ＤＹ－７５０、ＤＹ－７５１、ＤＹ－７５２、ＤＹ－７５４、ＤＹ－７７６、ＤＹ－７７７、ＤＹ－７７８、ＤＹ－７８０、ＤＹ－７８１、ＤＹ－７８２、ＤＹ－８００、ＤＹ－８３１（ＤＹＯＭＩＣＳ社製）、ＣＡＬ　Ｆｌｕｏｒ　Ｇｒｅｅｎ　５２０、ＣＡＬ　Ｆｌｕｏｒ　Ｇｏｌｄ　５４０、ＣＡＬ　Ｆｌｕｏｒ　Ｏｒａｎｇｅ　５６０、ＣＡＬ　Ｆｌｕｏｒ　Ｒｅｄ　５９０、ＣＡＬ　Ｆｌｕｏｒ　Ｒｅｄ　６１０、ＣＡＬ　Ｆｌｕｏｒ　Ｒｅｄ　６１５、ＣＡＬ　Ｆｌｕｏｒ　Ｒｅｄ　６３５、Ｐｕｌｓａｒ　６５０（登録商標、ＢｉｏＳｅａｒｃｈ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）、５，１０，１５，２０－テトラフェニルポルフィンテトラスルホン酸、亜鉛５，１０，１５，２０－テトラフェニルポルフィンテトラスルホン酸、フタロシアニンテトラスルホン酸、亜鉛フタロシアニンテトラスルホン酸、Ｎ，　Ｎ－Ｂｉｓ－（２，６－ｄｉｉｓｏｐｒｏｐｙｌｐｈｅｎｙｌ）－１，６，７，１２－（４－ｔｅｒｔ－ｂｕｔｙｌｐｈｅｎｏｘｙ）－ｐｅｒｙｌｅｎ－３，４，９，１０－ｔｅｔｒａｃａｒｂｏｎａｃｉｄ　ｄｉｉｍｉｄｅ、Ｎ，Ｎ’－Ｂｉｓ（２，６－ｄｉｉｓｏｐｒｏｐｙｌｐｈｅｎｙｌ）－１，６，７，１２－ｔｅｔｒａｐｈｅｎｏｘｙｐｅｒｙｌｅｎｅ－３，４：９，１０－ｔｅｔｒａｃａｒｂｏｘｄｉｉｍｉｄｅ、Ｂｅｎｚｅｎｅｓｕｌｆｏｎｉｃ　ａｃｉｄ，　４，４’，４’’，４’’’－［（１，３，８，１０－ｔｅｔｒａｈｙｄｒｏ－１，３，８，１０－ｔｅｔｒａｏｘｏｐｅｒｙｌｏ［３，４－ｃｄ：９，１０－ｃ’ｄ’］ｄｉｐｙｒａｎ－５，６，１２，１３－ｔｅｔｒａｙｌ）ｔｅｔｒａｌｉｓ（ｏｘｙ）］ｔｅｔｒａｋｉｓ－などを用いることができる。これら蛍光色素は単独で使用されてもよいし、複数種が混合され使用されてもよい。なお、このような色素分子の総称は、化合物中の主要な構造（骨格）または登録商標に基づき命名されており、それぞれに属する蛍光色素の範囲は当業者であれば過度の試行錯誤を要することなく適切に把握できるものである。蛍光色素が母体に内包されている場合、蛍光色素は母体内部に分散されていればよく、母体自体と化学的に結合していてもよいし、していなくてもよい。 Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2', 4,4', 5', 7,7'-hexachlorofluorescein, 6 -Carboxy-2', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy -Rhodamine, 5,6-dicarboxy-Rhodamine, Rhodamine 6G, Tetramethyl Rhodamine, X-Rhodamine, Sulfoldamine B, Sulfolodamine 101, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. , Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor6 Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 601 / / 665 (manufactured by Invitrogen), methoxykumarin, kumarin 6, kumarin 7, sulfokumarin 6, sulfokumarin 7, eodin, NBD, pyrene, Cy5, Cy5.5, Cy7, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluorescein 594, HiLite Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750 (registered trademark, manufactured by Anaspec), DyLight 350, DyLight 405, DyLight 488, DyLight 580, DyLight 550, DyLight 550, DyLight6 yLight 680, DyLight 755, DyLight 800 (registered trademark, manufactured by Thermo Scientific), ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rh ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO , ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740 (registered trademark, manufactured by ATTO-TEC), MFP488, MFP555, MFP590, MFP631 (registered trademark, manufactured by Mobitec), CF350, CF405S, CF405M , CF488A, CF514, CF532, CF543, CF555, CF568, CF594, CF620R, CF633, CF640R, CF647, CF660C, CF660R, CF680, CF680R, CF750, CF770, CF790 (registered trademark, Biotium), DY-350 -405, DY-415, DY-480XL, DY-481XL, DY-485XL, DY-490, DY-495, DY-505, DY-500XL, DY-510XL, DY-520XL, DY-521XL, DY-530 , DY-547P1, DY-549P1, DY-550, DY-554, DY-555, DY-556, DY-560, DY-590, DY-591, DY-594, DY-605, DY-610, DY -615, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, DY-647P1, DY-648P1, DY-649P1, DY-650, DY-654 , DY-651, DY-652, DY-675, DY-676, DY-677, DY-678, DY-679P1, DY-680, DY-681, DY-682, DY-700, DY-701, DY -703, DY-704, DY-730, DY-731, DY- 732, DY-734, DY-749P1, DY-750, DY-751, DY-752, DY-754, DY-776, DY-777, DY-778, DY-780, DY-781, DY-782, DY-800, DY-831 (manufactured by DYOMICS), CAL Fluor Green 520, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 610, CAL Fluor (Registered trademark, manufactured by BioSearch Technologies), 5,10,15,20-tetraphenylporphintetrasulfonic acid, zinc 5,10,15,20-tetraphenylporphintetrasulfonic acid, phthalocyaninetetrasulfonic acid, zinc phthalocyaninetetrasulfonic acid Acid, N, N-Bis- (2,6-diisopropylphenyl) -1,6,7,12- (4-tert-butylphenoxy) -perylen-3,4,9,10-terracarbonacid dimide, N, N'- Bis (2,6-diisopropylphenyl) -1,6,7,12-teraphenoxyperylene-3,4: 9,10-terracarboxdiimide, Benzenesulfonic acid, 4,4', 4'', 4'''-[(1,6) 3,8,10-tellahydro-1,3,8,10-tetraoxoperylo [3,4-cd: 9,10-c'd'] dipyran-5,6,12,13-tetrayl) terralis (oxy)] Tetracis- and the like can be used. These fluorescent dyes may be used alone or in combination of two or more. It should be noted that such dye molecules are generically named based on the main structure (skeleton) in the compound or a registered trademark, and the range of fluorescent dyes belonging to each requires excessive trial and error by those skilled in the art. It can be grasped properly without any problem. When the fluorescent dye is encapsulated in the mother body, the fluorescent dye may or may not be chemically bonded to the mother body itself as long as it is dispersed inside the mother body.
　本明細書において使用される場合、「生体マーカー分子」とは、ある状態（例えば、疾患、障害など）に罹患しているかまたはその危険性があるかどうかを追跡する示標となる物質をいう。このようなマーカーとしては、遺伝子、遺伝子産物、代謝物質、酵素などを挙げることができる。本発明において、生体マーカー分子としては、ＬＤＬ、ＡＧＥｓ、またはその類似物が挙げられる。生体マーカー分子には、健常状態と健常状態以外とで異なるレベルまたは形態のものがあり、健常状態以外において特徴的に見出されるも）を本明細書で特に「生体マーカー分子の異常型」という。生体マーカー分子の異常型は、疾患に特に関連が強いことから、本発明において検出対象とされ得る。生体マーカー分子の異常型は、健常者において通常観察されない、または観察されても少ない生体マーカーの修飾物であることが多い。生体マーカー分子の異常型としては、変性ＬＤＬおよび刺激性ＡＧＥｓなどが挙げられる。 As used herein, a "biological marker molecule" is a substance that serves as a marker for tracking whether a condition (eg, disease, disorder, etc.) is present or at risk. .. Examples of such markers include genes, gene products, metabolites, enzymes and the like. In the present invention, examples of the biomarker molecule include LDL, AGEs, and analogs thereof. Some biological marker molecules have different levels or morphologies in the healthy state and non-healthy states, and are characteristically found in other than the healthy state), which are particularly referred to herein as "abnormal forms of the biological marker molecule". Abnormal forms of biomarker molecules can be detected in the present invention because they are particularly strongly associated with disease. Abnormal forms of biomarker molecules are often modifications of biomarkers that are not normally observed or are rarely observed in healthy individuals. Abnormal forms of biomarker molecules include denatured LDL and stimulant AGEs.
　本明細書において「競合分子」とは、ある対象に対して結合する結合分子と、競合して結合する分子をいう。競合分子は、ある対象に対する結合が結合分子と競合するため、競合分子が存在すると、その結合分子の結合量が減少することから、競合分子を用いることで、その結合分子や結合（例えば、コンジュゲート形成）の量を間接的に測定することができる。 In the present specification, the "competitive molecule" means a binding molecule that binds to a certain object and a molecule that competes and binds to a certain object. Since the binding of a competing molecule to a certain object competes with the binding molecule, the amount of the binding molecule bound to the competing molecule decreases when the competing molecule exists. Therefore, by using the competing molecule, the binding molecule or bond (for example, Conju) can be used. The amount of gate formation) can be measured indirectly.
　本明細書において「コンジュゲート」とは、ある対象と別の実体とが結合して一体化することをいい、その能力を「コンジュゲート形成能」という。例えば、変性ＬＤＬについては、ＣＬＴＤ１４が該当し、刺激性ＡＧＥｓについては、ｓＲＡＧＥが該当し、他の分子についても当業者は適宜特定することができる。 In the present specification, "conjugate" means that a certain object and another entity are combined and integrated, and the ability thereof is referred to as "conjugate forming ability". For example, CLTD14 corresponds to denatured LDL, sRAGE corresponds to stimulating AGEs, and other molecules can be appropriately specified by those skilled in the art.
　本明細書において使用される場合、「検出用結合剤」とは、本開示のデバイスまたはキットにおけるメンブレンの検出部（例えば、テストスポットまたはライン）において、検出対象である生体マーカー分子もしくはその異常型、またはこれらの競合分子に特異的に結合する分子（例えば、抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメント、あるいは、抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメント等）を指す。 As used herein, a "detecting binder" is a biomarker molecule or anomalous form thereof to be detected at a detection site (eg, test spot or line) of a membrane in the devices or kits of the present disclosure. , Or a molecule that specifically binds to these competing molecules (eg, anti-LDL antibody, anti-modified LDL antibody or anti-ApoB antibody or antigen-binding fragment thereof, or anti-BSA antibody or anti-OVA antibody or antigen-binding fragment thereof, etc. ).
　本明細書において使用される場合、「コントロール用結合剤」とは、本開示のデバイスまたはキットにおけるメンブレンのコントロール部（例えば、コントロールスポットまたはライン）において、生体マーカー分子の異常型とコンジュゲート形成能を有する結合分子または生体マーカー分子の異常型の競合分子とコンジュゲート形成能を有する結合分子に特異的に結合する分子を指す。１つの実施形態では、コントロール用結合剤は、生体マーカー分子の異常型（例えば、変性ＬＤＬ）に特異的に結合する分子とコンジュゲート形成能を有する結合分子（例えば、ＣＴＬＤ１４）であるか、または生体マーカー分子（例えば、刺激性ＡＧＥｓなど）と競合する競合分子（例えば、Ｇ－ＢＳＡなど）とコンジュゲート形成能を有する結合分子（ｓＲＡＧＥ）であり得、生体マーカー分子の異常型が特定されれば、当業者は適宜、その分子とコンジュゲート形成能を有する結合分子（例えば、変性ＬＤＬの場合はＣＴＬＤ１４）と特異的に結合する分子（例えば、変性ＬＤＬの場合は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメント）を特定または生成し、あるいはその競合分子（例えば、刺激性ＡＧＥｓの場合はＧ－ＢＳＡもしくはＧ－ＯＶＡ）と特異的に結合する分子（例えば、刺激性ＡＧＥｓの場合は抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメント）を特定または生成することができ、これらをコントロール用結合剤として用いることができる。 As used herein, a "control binder" is an atypical form of a biomarker molecule and the ability to form a conjugate in a control section (eg, control spot or line) of a membrane in a device or kit of the present disclosure. Refers to a molecule that specifically binds to a binding molecule that has the ability to form a conjugate with an atypical competing molecule of a binding molecule or a biological marker molecule. In one embodiment, the control binding agent is a binding molecule (eg, CTLD14) capable of forming a conjugate with a molecule that specifically binds to an aberrant form of the biomarker molecule (eg, modified LDL), or It can be a binding molecule (sRAGE) capable of forming a conjugate with a competing molecule (eg, G-BSA) that competes with the biomarker molecule (eg, stimulating AGEs), and an abnormal type of the biomarker molecule has been identified. For example, those skilled in the art may appropriately bind to a binding molecule having the ability to form a conjugate with the molecule (for example, CTLD14 in the case of modified LDL) (for example, anti-LDL antibody in the case of modified LDL, anti-modified LDL). A molecule that identifies or produces an antibody or anti-ApoB antibody or an antigen-binding fragment thereof, or specifically binds to a competing molecule thereof (eg, G-BSA or G-OVA in the case of stimulating AGEs) (eg, stimulus). In the case of sex AGEs, an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof) can be identified or produced, and these can be used as a control binding agent.
　本明細書において「メンブレン」とは多孔性または非多孔性の水に不溶性の材料である固相を指し、好ましくは生体分子を結合又は保持し得る能力を有する材料を少なくとも部分的に有するものであり得る。本明細書において用いられるメンブレンは、好ましくは毛管現象による試料の展開を行うことができるものであり、それらを実現する材料を少なくとも一部含み得る。メンブレンを構成する材料の限定的な例として、例えば、セルロース、ＳＥＰＨＡＤＥＸ（商標）などの多糖、ガラス、ポリアクリロイルモルホリド、シリカ、コントロールドポア（ｃｏｎｔｒｏｌｌｅｄ　ｐｏｒｅ）ガラス（ＣＰＧ）、ポリスチレン、ポリスチレン／ラテックス、超高分子量ポリエチレン（ＵＰＥ）などのポリエチレン、ポリアミド、ポリビニリジンフルオリド（ＰＶＤＦ）、ポリテトラフルオロエチレン（ＰＴＦＥ；テフロン（登録商標））、カルボキシル修飾テフロン（登録商標）、ナイロン、ニトロセルロースならびに金、白金およびパラジウムなどの金属および合金で構成され得る。通常、メンブレンは、帯電しており、タンパク質などの有機材料と結合する。メンブレンは、プロセスを定量的にすることによって、各種分析プロセスを大幅に改善する。 As used herein, the term "membrane" refers to a solid phase that is a porous or non-porous water-insoluble material, preferably one that at least partially has a material capable of binding or retaining biomolecules. possible. The membrane used in the present specification is preferably capable of developing a sample by capillarity, and may contain at least a part of a material for realizing them. Limited examples of materials that make up the membrane include, for example, cellulose, polysaccharides such as Sephadex ™, glass, polyacryloyl morpholide, silica, controlled pore glass (CPG), polystyrene, polystyrene / Polyethylene such as latex, ultra-high molecular weight polyethylene (UPE), polyamide, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE; Teflon®), carboxyl-modified Teflon®, nylon, nitrocellulose And can be composed of metals and alloys such as gold, platinum and palladium. Membranes are usually charged and bind to organic materials such as proteins. Membranes significantly improve various analytical processes by making the process quantitative.
　本明細書において使用される場合、「変性ＬＤＬ」は、「ＬＤＬ修飾物」「修飾ＬＤＬ」（これらは、同じ意味で用いられる）とも呼ばれ、ＬＤＬが体内で活性酸素、酸化的酵素、Ｆｅ３＋などと接触すること、あるいは、血管内皮細胞やマクロファージなどによる細胞依存性化学変化によって発生する種々の分子修飾を有する任意のＬＤＬ修飾物である。生体内に存在するＬＤＬ修飾物としては、代表的には、酸化ＬＤＬ（本明細書中、ＯｘＬＤＬといい、例としては完全酸化ＬＤＬ（本明細書中ｆｕ　ＯｘＬＤＬともいう）および部分酸化ＬＤＬ（本明細書中ｍｏ　ＯｘＬＤＬともいう）が挙げられる）、マロンジアルデヒド化ＬＤＬ（ＭＤＡ－ＬＤＬ）、クロトンアルデヒド（ＣＲＡ）修飾ＬＤＬ、等のアルデヒド修飾ＬＤＬ、アクロレイン修飾ＬＤＬ、ノネナール修飾ＬＤＬ、４－ヒドロキシノネナール（ＨＮＥ）修飾ＬＤＬ、ヘキサノイル（ＨＥＬ）修飾ＬＤＬ、小粒子ＬＤＬ（直径２５５ｎｍ以下のＬＤＬ）、糖化ＬＤＬ、アセチル化ＬＤＬ（ＡｃＬＤＬ）などが挙げられるが、これらに限定されない。酸化ＬＤＬが異常値を示す場合、動脈硬化症、虚血性心疾患（心筋梗塞、狭心症など）、脳血管障害（脳梗塞、脳出血、くも膜下出血、一過性脳虚血発作など）、大動脈瘤、腎梗塞、高脂血症などのような疾患が予想されるがこれらに限定されない（「今日の臨床検査２００７－２００８」発行所株式会社南江堂、参照）。一般に使用される検査方法では、基準物質としては、ＭＤＡ－ＬＤＬ（正常範囲：１０～８０ΜＬ）および酸化ホスファチジルコリン（正常範囲：８．４Ｕ／ｍＬ～１７．６Ｕ／ｍＬ）が使用されている。 As used herein, "modified LDL" is also referred to as "LDL modified product" and "modified LDL" (these are used interchangeably), and LDL is an active oxygen species, oxidative enzyme, Fe3 + in the body. It is any LDL-modified product having various molecular modifications generated by contact with such as, or by cell-dependent chemical changes caused by vascular endothelial cells, macrophages, and the like. Typical LDL modifications existing in the living body include oxidized LDL (referred to as OxLDL in the present specification, and for example, fully oxidized LDL (also referred to as fu OxLDL in the present specification) and partially oxidized LDL (the present specification). (Also referred to as mo OxLDL in the specification)), malondialdehyde-modified LDL (MDA-LDL), crotonaldehyde (CRA) -modified LDL, and other aldehyde-modified LDL, achlorine-modified LDL, nonenal-modified LDL, 4-hydroxynonenal. (HNE) modified LDL, hexanoyl (HEL) modified LDL, small particle LDL (LDL having a diameter of 255 nm or less), saccharified LDL, acetylated LDL (AcLDL) and the like can be mentioned, but are not limited thereto. If the oxidized LDL shows abnormal values, arteriosclerosis, ischemic heart disease (myocardial infarction, angina, etc.), cerebrovascular accident (cerebral infarction, cerebral bleeding, submucosal bleeding, transient ischemic attack, etc.), Diseases such as aortic aneurysm, renal infarction, and hyperlipidemia are expected, but not limited to these (see "Today's Clinical Examination 2007-2008" Publisher Nanedo Co., Ltd.). In commonly used inspection methods, MDA-LDL (normal range: 10 to 80 ΜL) and oxidized phosphatidylcholine (normal range: 8.4 U / mL to 17.6 U / mL) are used as reference substances.
　本明細書において使用される場合、用語「ＣＴＬＤ分子」とは、ＣＴＬＤ様ポリペプチドの他、それらの任意の複合体を含むことが理解される。したがって、ＣＴＬＤ分子には、ＬＯＸ－１全長、ＬＯＸ－１細胞外領域全長（Ｓ６１～Ｑ２７３）、ＣＴＬＤ１４（１２９－１４３）、ＣＴＬＤ（１４３－２７３）等も包含されることが理解される。本明細書において使用される場合、用語「ＣＴＬＤ１４」および「ＰＲ－ＣＴＬＤ１４」とは、（１）配列番号２に示されるアミノ酸配列からなるポリペプチド；（２）上記配列番号２に示されるアミノ酸配列において１または数個のアミノ酸の置換、付加および／または欠失を含むアミノ酸配列を含むポリペプチド；（３）上記配列番号２に示されるアミノ酸配列において１０４位および１２１位以外のアミノ酸位置で１または数個の置換、付加および／または欠失を含み、かつ天然型ＬＯＸ－１の活性を示すアミノ酸配列を含むポリペプチド；（４）上記配列番号２に示されるアミノ酸配列と少なくとも９０％の配列同一性を有するアミノ酸配列を含むポリペプチド；（５）上記配列番号２に示されるアミノ酸配列と少なくとも８０％の配列相同性を有するアミノ酸配列を含むポリペプチド；（６）配列番号１に示される核酸分子によってコードされるアミノ酸配列を含むポリペプチド；（７）上記配列番号１に示される核酸配列に対して相補的な核酸配列とストリンジェントな条件下でハイブリダイズする核酸分子によってコードされるアミノ酸配列を含むポリペプチド；（８）配列番号１に示される核酸配列と相補的な核酸配列とストリンジェントな条件下でハイブリダイズする核酸分子によってコードされるアミノ酸配列であって、該コードされるアミノ酸配列において１０４位および１２１位のアミノ酸は、配列番号２における対応するアミノ酸を保持し、かつ天然型ＬＯＸ－１の活性を示すアミノ酸配列を含むポリペプチド；（９）上記配列番号１に示される核酸配列において１または数個の置換、付加および／または欠失を有する核酸配列によってコードされるアミノ酸配列を含み、かつ天然型ＬＯＸ－１の活性を示すポリペプチド；（１０）上記配列番号１に示される核酸配列と少なくとも９０％の配列同一性を有する核酸配列によってコードされるアミノ酸配列を含むポリペプチド；（１１）上記配列番号１に示される核酸配列と少なくとも８０％の配列相同性を有する核酸配列によってコードされるアミノ酸配列を含むポリペプチド、のうちの１つによって示される。上記の同一性または相同性は、配列分析用ツールであるＢＬＡＳＴ（ＮＣＢＩのＢＬＡＳＴ　２．９（２０１９．３．１１　発行））を用いてデフォルトパラメータを用いて算出される。ストリンジェントな条件は配列に依存して変化し、このような条件の決定は、当業者の技術範囲内である。 As used herein, the term "CTLD molecule" is understood to include CTLD-like polypeptides as well as any complex thereof. Therefore, it is understood that the CTLD molecule also includes the LOX-1 total length, the LOX-1 extracellular space total length (S61 to Q273), CTLD14 (129-143), CTLD (143-273), and the like. As used herein, the terms "CTLD14" and "PR-CTLD14" are: (1) a polypeptide consisting of the nucleic acid sequence set forth in SEQ ID NO: 2; (2) the amino acid sequence set forth in SEQ ID NO: 2 above. Polypeptide containing an amino acid sequence containing substitutions, additions and / or deletions of one or several amino acids in; (3) 1 or 1 or 1 at an amino acid position other than positions 104 and 121 in the amino acid sequence shown in SEQ ID NO: 2 above. A polypeptide containing several substitutions, additions and / or deletions and containing an amino acid sequence exhibiting the activity of native LOX-1; (4) At least 90% sequence identical to the amino acid sequence shown in SEQ ID NO: 2 above. A polypeptide containing a sex amino acid sequence; (5) a polypeptide containing an amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 2 above; (6) a nucleic acid molecule shown in SEQ ID NO: 1. Polypeptide containing an amino acid sequence encoded by; (7) An amino acid sequence encoded by a nucleic acid molecule that hybridizes with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 1 above under stringent conditions. Polypeptide containing; (8) An amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 1, and in the encoded amino acid sequence. The 104th and 121st amino acids are polypeptides that retain the corresponding amino acid in SEQ ID NO: 2 and contain an amino acid sequence exhibiting the activity of native LOX-1; (9) in the nucleic acid sequence shown in SEQ ID NO: 1 above. A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having one or several substitutions, additions and / or deletions and exhibiting the activity of native LOX-1; (10) Nucleic acid set forth in SEQ ID NO: 1 above. Polypeptide containing an amino acid sequence encoded by a nucleic acid sequence having at least 90% sequence identity with the sequence; (11) encoded by a nucleic acid sequence having at least 80% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 1 above. It is indicated by one of the polypeptide comprising the amino acid sequence to be made. The above identity or homology is calculated using default parameters using BLAST (NCBI BLAST 2.9 (issued March 11, 2019)), which is a tool for sequence analysis. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of one of ordinary skill in the art.
　本明細書において使用される場合、用語「終末糖化産物」または「後期糖化反応生成物」（いずれも英文では、Ａｄｖａｎｃｅｄ　Ｇｌｙｃａｔｉｏｎ　Ｅｎｄ　Ｐｒｏｄｕｃｔｓ）とは、ＡＧＥｓともいわれ、タンパク質の糖化産物であり多様な構造体の総称である。食品の加工過程で生じ、食味向上に重要である一方、生体内でも生成し、一部は生体に機能不全を誘導し加齢性疾患の引き金となる。糖尿病患者の生活の質を損ねる元凶である血管合併症として知られる糖尿病性血管障害の発症・進展への関与も知られている。血管合併症による眼、神経、腎臓の障害は、それぞれ糖尿病網膜症、神経症、腎症（あわせて三大合併症）とよばれており、糖尿病患者に特徴的な病態である。グルコースに代表される還元糖は、タンパク質、アミノ酸のアミノ基と非酵素的に反応して、シッフ塩基またはアマドリ転位化合物などの糖化生成物を形成する。ここまでの反応は可逆的であり、前期反応とよばれている。その後、さらに縮合、開裂、架橋形成などの複雑かつ不可逆的な反応を経て、終末糖化産物を形成する。このような一連の反応は、糖化反応と称される。ＡＧＥｓはまた、このような過程を経て生成された構造物の総称である。生体中に存在するＡＧＥｓ構造としては、カルボキシメチルリジン(ＣＭＬ)、カルボキエチルリジン(ＣＥＬ)、ペントシジン、ピラリン、イミダゾリン、メチルグリオキサール、クロスリンなどが挙げられるが、これに限定されない。血漿中に存在するアルブミン、イムノグロブリン、オボアルブミンなどが上記の糖化を受けた産物もＡＧＥであり、ＡＧＥとして実験系に汎用されている。さらに、インビトロ実験系では、ＢＳＡ（ウシ血清アルブミン）に糖化処理を施したもの、例えば、Ｒ－ＡＧＥ（リボースにより糖化処理をしたＢＳＡ）、Ｆ－ＡＧＥ（フルクトースにより処理をしたＢＳＡ）；Ｇ－ＡＧＥ（グルコースにより糖化処理をしたＢＳＡ）なども汎用されている。血糖コントロールの指標として用いられているヘモグロビンＡ１ｃはアマドリ転移化合物であるが、ＡＧＥｓに包含される。また、任意のタンパク質も、ＡＧＥｓに変換可能である。例えば、ＡＧＥｓに包含されるＣＭＬアルブミンおよびＣＥＬアルブミンは、いずれもアルブミンが糖化を受けたＡＧＥｓである。このようなＡＧＥｓ生成反応は、生体内において循環血液中、細胞外マトリクス、細胞内のいずれでも起こり得る。例えば、糖尿病患者の血管に存在するＡＧＥｓとしては、：蛍光性で架橋構造を有するもの（ペントシジン、クロスリンなど）および蛍光も架橋もないもの（カルボキシメチルリジン、ピラリン、メチルグリオキサール（ＭＧ）－イミダゾロンなど）の２つに大別できる。ＡＧＥｓが異常値を示す場合、細小血管症（腎症、網膜症、神経症など）、大血管障害（虚血性心疾患、脳血管疾患、閉塞性動脈硬化症のような疾患が予想される。一般に使用される検査方法では、基準物質としては、ピラリン（正常範囲：血漿中２３ｐｍｏｌ／ｍＬ未満）、ペントシジン（正常範囲：血漿中０．００９１５～０．０４３１μｇ／ｍＬ（ＥＬＩＳＡで測定した場合））などが使用される（「今日の臨床検査　２００７－２００８」発行所　株式会社　南江堂、参照）。 As used herein, the terms "advanced glycation end products" or "advanced glycation end products" (both in English, Advanced Glycation End Products) are also referred to as AGEs, which are glycation products of proteins and have various structures. It is a general term for the body. It occurs in the process of processing foods and is important for improving the taste, but it is also produced in the living body, and a part of it induces dysfunction in the living body and triggers age-related diseases. It is also known to be involved in the onset and progression of diabetic angiopathy known as vascular complications, which is the main cause of impairing the quality of life of diabetic patients. Damage to the eyes, nerves, and kidneys due to vascular complications is called diabetic retinopathy, neuropathy, and nephropathy (total of the three major complications), and is a pathological condition characteristic of diabetic patients. Reducing sugars such as glucose react non-enzymatically with amino groups of proteins and amino acids to form glycation products such as Schiff bases or Amadori rearrangement compounds. The reaction up to this point is reversible and is called the early reaction. After that, advanced glycation end products are formed through complicated and irreversible reactions such as condensation, cleavage, and crosslink formation. Such a series of reactions is called a saccharification reaction. AGEs are also a general term for structures produced through such a process. Examples of the AGEs structure present in the living body include, but are not limited to, carboxymethyl lysine (CML), carboxyethyl lysine (CEL), pentosidine, pyrarin, imidazoline, methylglyoxal, and crosulin. The product in which albumin, immunoglobulin, ovalbumin and the like present in plasma undergo the above-mentioned saccharification is also AGE, and is widely used in experimental systems as AGE. Furthermore, in the in vitro experimental system, BSA (bovine serum albumin) that has been glycated, for example, R-AGE (BSA that has been glycated with ribose), F-AGE (BSA that has been treated with fructose); G- AGE (BSA saccharified with glucose) and the like are also widely used. Hemoglobin A1c, which is used as an index of glycemic control, is an Amadori rearrangement compound, but is included in AGEs. Also, any protein can be converted to AGEs. For example, CML albumin and CEL albumin included in AGEs are both AGEs in which albumin is glycated. Such an AGEs production reaction can occur in the circulating blood, extracellular matrix, or intracellular in vivo. For example, AGEs present in the blood vessels of diabetic patients include: fluorescent and cross-linked structures (pentosidine, crosulin, etc.) and non-fluorescent and non-cross-linked (carboxymethyl lysine, pyrarin, methylglyoxal (MG) -imidazolone, etc.) ) Can be roughly divided into two. When AGEs show abnormal values, diseases such as microangiopathy (nephropathy, retinopathy, neuropathy, etc.) and macroangiopathy (ischemic heart disease, cerebrovascular disease, arteriosclerosis obliterans, etc.) are expected. In commonly used test methods, the reference substances are pyrarin (normal range: less than 23 pmol / mL in plasma) and pentocidin (normal range: 0.00915 to 0.0431 μg / mL in plasma (as measured by ELISA)). Etc. are used (see "Today's Clinical Examination 2007-2008" Publisher, Nanedo Co., Ltd.).
　本明細書において、「刺激性の終末糖化産物」または「刺激性のＡＧＥｓ（刺激性ＡＧＥｓ）」とは、疾患との関連性が高いＡＧＥｓを指し、ｓＲＡＧＥと強く結合する性質を有している。以前は、血中のグルコースによる糖化が主であると考えられていたが、グルコースによる糖化は長時間を要する上、グルコース糖化ＡＧＥｓは生体への刺激性も弱いことが示唆され始めた。過剰なグルコースは、ポリオール代謝系で代謝されグリセルアルデヒド（Ｇｌｙｃｅｒ）を生じるほか、酸化反応によりグリオキサール（ＧＯ）、グリコールアルデヒド（Ｇｌｙｃｏｌ）などを生じるが、これらは、反応性が高く、短時間でＡＧＥｓを生成する上、その糖化産物は生体刺激性が高いことが報告されている。肝疾患（例えば、ＮＡＳＨ）は、特にグリセルアルデヒドによる修飾を受けたタンパク質（Ｇｌｙｃｅｒ－ＡＧＥｓ）と関連性が高いことが示唆されている。 In the present specification, "stimulated advanced glycation end products" or "stimulated AGEs (stimulated AGEs)" refer to AGEs that are highly related to diseases and have a property of strongly binding to sRAGE. .. Previously, it was thought that glycation by glucose in blood was the main cause, but glycation by glucose takes a long time, and it has begun to be suggested that glucose glycated AGEs are less irritating to the living body. Excess glucose is metabolized in the polyol metabolism system to produce glyceraldehyde (Glycer), and the oxidation reaction produces glyoxal (GO), glycolaldehyde (Glycol), etc., which are highly reactive and can be produced in a short time. In addition to producing AGEs, it has been reported that the glycated product is highly biostimulant. It has been suggested that liver disease (eg, NASH) is particularly associated with proteins modified with glyceraldehyde (Glycer-AGEs).
　本明細書において「ＡＧＥｓ分子」とは、上記ＡＧＥに含まれる任意の分子をいう。例えば、ＡＧＥｓとしては、Ｌｙｓ－ＡＧＥ（グルタルアルデヒド修飾リジン修飾ＡＧＥ）、グルコース修飾ＡＧＥ（Ｇ－ＡＧＥ）、リボース修飾ＡＧＥ（Ｒ－ＡＧＥ）、フルクトース修飾ＡＧＥ（Ｆ－ＡＧＥ）またはそれらの改変体あるいはそれらの複合体を挙げることができるがそれらに限定されない。 In the present specification, the "AGEs molecule" means any molecule contained in the above-mentioned AGE. For example, AGEs include Lys-AGE (glutaraldehyde-modified lysine-modified AGE), glucose-modified AGE (G-AGE), ribose-modified AGE (R-AGE), fructose-modified AGE (F-AGE), or a variant thereof. Complexes thereof can be mentioned, but are not limited to them.
　本明細書において「ＡＧＥｓ様活性を示す分子」とは、少なくとも上述のＡＧＥｓの活性（本明細書において「ＡＧＥｓ様活性」という。）の一つを有する分子をいう。そのようなＡＧＥｓ様活性としては、ＲＡＧＥに対する結合活性（リガンド活性）を挙げることができるが、それに限定されない。 In the present specification, the "molecule exhibiting AGEs-like activity" refers to a molecule having at least one of the above-mentioned activities of AGEs (referred to as "AGEs-like activity" in the present specification). Examples of such AGEs-like activity include, but are not limited to, binding activity to RAGE (ligand activity).
　本明細書において使用される場合、用語「ＡＧＥ受容体（Ｒｅｃｅｐｔｏｒ　ｆｏｒ　ＡＧＥ）」とは、ＲＡＧＥともいわれ、（１）配列番号４に示されるアミノ酸配列からなるポリペプチド；（２）上記配列番号４に示されるアミノ酸配列において１または数個のアミノ酸の置換、付加および／または欠失を含むアミノ酸配列を含み、かつ天然型ＲＡＧＥの活性を示すポリペプチド；（３）上記配列番号４に示されるアミノ酸配列と少なくとも９０％の配列同一性を有するアミノ酸配列を含み、かつ天然型ＲＡＧＥの活性を示すポリペプチド；（４）上記配列番号４に示されるアミノ酸配列と少なくとも８０％の配列相同性を有するアミノ酸配列を含み、かつ天然型ＲＡＧＥの活性を示すポリペプチド；（５）配列番号３に示される核酸分子によってコードされるアミノ酸配列を含むポリペプチド；（６）上記配列番号３に示される核酸配列に対して相補的な核酸配列とストリンジェントな条件下でハイブリダイズする核酸分子によってコードされるアミノ酸配列を含み、かつ天然型ＲＡＧＥの活性を示すポリペプチド；（７）上記配列番号３に示される核酸配列において１または数個のヌクレオチドの置換、付加および／または欠失を有する核酸分子によってコードされるアミノ酸配列を含み、かつ天然型ＲＡＧＥの活性を示すポリペプチド；（８）上記配列番号３に示される核酸配列と少なくとも９０％の配列同一性を有する核酸分子によってコードされるアミノ酸配列を含み、かつ天然型ＲＡＧＥの活性を示すポリペプチド；および（９）上記配列番号３に示される核酸配列と少なくとも８０％の配列相同性を有する核酸分子によってコードされるアミノ酸配列を含み、かつ天然型ＲＡＧＥの活性を示すポリペプチド、のうちの１つである。上記の同一性または相同性は、配列分析用ツールであるＢＬＡＳＴ（ＮＣＢＩのＢＬＡＳＴ　２．９（２０１９．３．１１　発行））を用いてデフォルトパラメータを用いて算出される。ストリンジェントな条件は配列に依存して変化し、このような条件の決定は、当業者の技術範囲内である。ＲＡＧＥはまた、１９９２年に、ウシ肺から同定され、ＡＧＥと結合するイムノグロブリンスーパーファミリーに属する、分子量約３５ｋＤａのＩ型膜タンパク質（糖鎖修飾を受けた完全なＲＡＧＥは、分子量５５ｋＤａ）である。ＲＡＧＥｓの細胞外ドメインは、１つのＶ型イムノグロブリンドメイン、続いて、２つのＣ型イムノグロブリンドメイン（Ｃ１領域およびＣ２領域）の、３つのイムノグロブリンフォールド構造を取るドメインが結合した構造を取っている。ＲＡＧＥはまた、細胞膜１回貫通型のドメインおよび４３アミノ酸の細胞質ドメインを含む。ＲＡＧＥは、多様なクラスのリガンド（ＡＧＥｓ、Ｓ１００／カルグラニュリン、アンフォテリンおよびアミロイド－βペプチド）と相互作用する。Ｖドメインは、リガンド結合に必須の部位であり、細胞質ドメインは、ＲＡＧＥ媒介性細胞内シグナル伝達に必須である。ＲＡＧＥはまた、各ドメイン内でジスルフィド結合を有するので、本開示の変異ＲＡＧＥ－界面活性剤複合体は、配列番号６のアミノ酸配列における３８位、９９位、１４４位、２０８位、２５９位および３０１位に対応するシステイン残基を保持していることが好ましい。ＲＡＧＥは、正常組織および血管系においては低レベルでしか発現されない。しかし、この受容体は、そのリガンドが蓄積した場所においてアップレギュレートされる。ＲＡＧＥの発現は、糖尿病血管系において内皮細胞、平滑筋細胞、周皮細胞、腎メサンギウム細胞および浸潤性単核食細胞で増加している。また、ＡＧＥｓが蓄積している動脈硬化巣のような病的部位においても、ＲＡＧＥの発現が増加している。ＡＧＥｓ－ＲＡＧＥ相互作用は、血管系ホメオスタシスにおいて重要な細胞の特性を変化させる。例えば、ＲＡＧＥがＡＧＥｓと結合した後、血管内皮細胞は、ＶＣＡＭ－１、組織因子、およびＩＬ－６の発現、ならびに高分子へのそれらの透過性を増加させる。単核食細胞において、ＲＡＧＥは、サイトカインおよび増殖因子の発現を活性化し、可溶性ＡＧＥｓに応じて細胞移動を誘導するのに対して、走触性は、固定リガンドで起こる。 As used herein, the term "Receptor for AGE" is also referred to as RAGE, and is (1) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; (2) the above-mentioned SEQ ID NO: 4 A polypeptide containing an amino acid sequence containing one or several amino acid substitutions, additions and / or deletions in the nucleic acid sequence shown in the above, and exhibiting the activity of natural RAGE; (3) Nucleic acid shown in SEQ ID NO: 4 above. A polypeptide containing an amino acid sequence having at least 90% sequence identity with the sequence and exhibiting the activity of natural RAGE; (4) Nucleic acid having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 4 above. A polypeptide containing a sequence and exhibiting the activity of native RAGE; (5) a polypeptide containing an amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO: 3; (6) in the nucleic acid sequence shown in SEQ ID NO: 3 above. A polypeptide containing an amino acid sequence encoded by a nucleic acid molecule that hybridizes with a complementary nucleic acid sequence and a nucleic acid molecule that hybridizes under stringent conditions, and exhibits the activity of natural RAGE; (7) Nucleic acid shown in SEQ ID NO: 3 above. Polypeptide containing an amino acid sequence encoded by a nucleic acid molecule having one or several nucleotide substitutions, additions and / or deletions in the sequence and exhibiting the activity of native RAGE; (8) shown in SEQ ID NO: 3 above. A polypeptide containing an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity with the nucleic acid sequence and exhibiting the activity of native RAGE; and (9) at least the nucleic acid sequence shown in SEQ ID NO: 3 above. It is one of a polypeptide containing an amino acid sequence encoded by a nucleic acid molecule having 80% sequence homology and exhibiting the activity of native RAGE. The above identity or homology is calculated using default parameters using BLAST (NCBI BLAST 2.9 (issued March 11, 2019)), which is a tool for sequence analysis. Stringent conditions vary depending on the sequence, and determination of such conditions is within the skill of one of ordinary skill in the art. RAGE is also a type I membrane protein with a molecular weight of approximately 35 kDa (a complete RAGE with sugar chain modification has a molecular weight of 55 kDa), which was identified in bovine lung in 1992 and belongs to the immunoglobulin superfamily that binds to AGE. .. The extracellular domain of RAGEs has a structure in which one V-type immunoglobulin domain is followed by two C-type immunoglobulin domains (C1 region and C2 region), which have three immunoglobulin fold structures. There is. RAGE also contains a transmembrane domain and a 43 amino acid cytoplasmic domain. RAGE interacts with various classes of ligands (AGEs, S100 / calgranulin, amphoterin and amyloid-β peptide). The V domain is an essential site for ligand binding and the cytoplasmic domain is essential for RAGE-mediated intracellular signaling. Since RAGE also has disulfide bonds within each domain, the mutant RAGE-surfactant complex of the present disclosure is at positions 38, 99, 144, 208, 259 and 301 in the amino acid sequence of SEQ ID NO: 6. It preferably retains the cysteine residue corresponding to the position. RAGE is expressed only at low levels in normal tissues and the vascular system. However, this receptor is upregulated where the ligand accumulates. RAGE expression is increased in endothelial cells, smooth muscle cells, pericytes, renal mesangial cells and infiltrating mononuclear phagocytes in the diabetic vasculature. In addition, the expression of RAGE is also increased in pathological sites such as arteriosclerotic lesions in which AGEs are accumulated. AGEs-RAGE interactions alter important cellular properties in vascular homeostasis. For example, after RAGE binds to AGEs, vascular endothelial cells increase the expression of VCAM-1, tissue factor, and IL-6, as well as their permeability to macromolecules. In mononuclear phagocytes, RAGE activates cytokine and growth factor expression and induces cell migration in response to soluble AGEs, whereas haptotaxis occurs with fixed ligands.
　本明細書では、「ＲＡＧＥリガンド認識領域」または「ｓＲＡＧＥ（ｓｏｌｕｂｌｅ　Ｒｅｃｅｐｔｏｒｆｏｒ　Ａｄｖａｎｃｅｄ　Ｇｌｙｃａｔｉｏｎ　Ｅｎｄ　ｐｒｏｄｕｃｔｓ）」とは、交換可能に使用され、ＲＡＧＥリガンドが認識する領域をいう。詳細には、ｓＲＡＧＥすなわちＲＡＧＥリガンド認識領域は、ＲＡＧＥの細胞外領域の全部または一部をいう。ｓＲＡＧＥは、代表的には、配列番号６または配列番号４の２２～３３２位で構成されるがこれに限定されない。 In the present specification, the "RAGE ligand recognition region" or "sRAGE (Soluble Receptor Advanced Glycation End products)" is used interchangeably and refers to a region recognized by the RAGE ligand. Specifically, the sRAGE or RAGE ligand recognition region refers to all or part of the extracellular space of RAGE. The sRAGE is typically composed of, but not limited to, positions 22-332 of SEQ ID NO: 6 or SEQ ID NO: 4.
　本明細書において使用される場合、用語「ＲＡＧＥ様ポリペプチド」とは、「ＲＡＧＥ８」、「ｍＲＡＧＥ８」、「ＲＡＧＥ１」、「ｍＲＡＧＥ１」、「ＲＡＧＥ２」、「ｍＲＡＧＥ２」、「ＲＡＧＥ３」、「ｍＲＡＧＥ３」、「ＲＡＧＥ４」、「ｍＲＡＧＥ４」、「ＲＡＧＥ７」、「ｍＲＡＧＥ７」、「ＲＡＧＥ１４３」、「ｍＲＡＧＥ１４３」、「ＲＡＧＥ２２３」、「ｍＲＡＧＥ２２３」、「ＲＡＧＥ２２６」および「ｍＲＡＧＥ２２６」と称されるポリペプチドまたはこれらの変異体を包含する。これらの説明は、特開２０１３－２０９３３０等に開示されており、適宜本明細書において参考としてその内容を援用する。 As used herein, the term "RAGE-like polypeptide" means "RAGE8", "mRAGE8", "RAGE1", "mRAGE1", "RAGE2", "mRAGE2", "RAGE3", "mRAGE3". , "RAGE4", "mRAGE4", "RAGE7", "mRAGE7", "RAGE143", "mRAGE143", "RAGE223", "mRAGE223", "RAGE226" and "mRAGE226" or their variants. Including the body. These explanations are disclosed in Japanese Patent Application Laid-Open No. 2013-209330 and the like, and the contents thereof are appropriately incorporated herein by reference.
　本明細書において使用される場合、用語「ＲＡＧＥ分子」とは、ＲＡＧＥ様ポリペプチドの他、それらの任意の複合体を含むことが理解される。したがって、ＲＡＧＥ分子には、ＲＡＧＥ様ポリペプチド等、例えば、ＲＡＧＥ（全長）、ＲＡＧＥ細胞外領域（配列番号４の２２－３３２位）、ＲＡＧＥ１４３、ＲＡＧＥ２２３、ＲＡＧＥ２２６等が包含されることが理解される。また、ＲＡＧＥ分子は、ＲＡＧＥを構成する３つのドメインのうちあるドメイン全体、またはあるドメインの一部を欠いたＲＡＧＥ（ミニＲＡＧＥ）も包含される。また、ミニＲＡＧＥは、ＲＡＧＥ様ポリペプチドのミニＲＡＧＥも包含する。 As used herein, the term "RAGE molecule" is understood to include RAGE-like polypeptides as well as any complex thereof. Therefore, it is understood that the RAGE molecule includes RAGE-like polypeptides and the like, for example, RAGE (overall length), RAGE extracellular region (position 22-332 of SEQ ID NO: 4), RAGE143, RAGE223, RAGE226 and the like. .. The RAGE molecule also includes a RAGE (mini RAGE) lacking a whole domain or a part of a certain domain among the three domains constituting the RAGE. The mini-RAGE also includes a mini-RAGE of a RAGE-like polypeptide.
　本明細書では、「ＲＡＧＥリガンド認識領域」を含む分子は、「ＲＡＧＥ分子」のうち全長ＲＡＧＥ以外のもの（「ＲＡＧＥ様ポリペプチド」を含む）、例えば、「ＲＡＧＥ８」、「ｍＲＡＧＥ８」、「ＲＡＧＥ１」、「ｍＲＡＧＥ１」、「ＲＡＧＥ２」、「ｍＲＡＧＥ２」、「ＲＡＧＥ３」、「ｍＲＡＧＥ３」、「ＲＡＧＥ４」、「ｍＲＡＧＥ４」、「ＲＡＧＥ７」、「ｍＲＡＧＥ７」、「ＲＡＧＥ１４３」、「ｍＲＡＧＥ１４３」、「ＲＡＧＥ２２３」、「ｍＲＡＧＥ２２３」、「ＲＡＧＥ２２６」および「ｍＲＡＧＥ２２６」、ＲＡＧＥ細胞外領域（配列番号４の２２－３３２位）、などを挙げることができる。 In the present specification, the molecule containing the "RAGE ligand recognition region" is a "RAGE molecule" other than the full-length RAGE (including the "RAGE-like polypeptide"), for example, "RAGE8", "mRAGE8", "RAGE1". , "MRAGE1", "RAGE2", "mRAGE2", "RAGE3", "mRAGE3", "RAGE4", "mRAGE4", "RAGE7", "mRAGE7", "RAGE143", "mRAGE143", "RAGE223", Examples thereof include "mRAGE223", "RAGE226" and "mRAGE226", and the RAGE extracellular region (position 22-332 of SEQ ID NO: 4).
　なお、上記ＲＡＧＥ様ポリペプチドは、天然型ＲＡＧＥの活性が保持されている限り、非天然アミノ酸を含んでいてもよいし、アミノ酸アナログ、アミノ酸誘導体などを含んでいてもよい。 The RAGE-like polypeptide may contain an unnatural amino acid, an amino acid analog, an amino acid derivative, or the like as long as the activity of the natural RAGE is retained.
　上記のＲＡＧＥ様ポリペプチドにおいても、分子内ジスルフィド結合を形成することは重要であるので、配列番号４のアミノ酸配列の３８位、９９位、１４４位、２０８位、２５９位および３０１位に対応するシステインは保持されていることが好ましい。 Since it is important to form an intramolecular disulfide bond also in the above RAGE-like polypeptide, it corresponds to the 38th, 99th, 144th, 208th, 259th and 301st positions of the amino acid sequence of SEQ ID NO: 4. Cysteine is preferably retained.
　本明細書において、「リガンド」とは、特異的な受容体または受容体のファミリーに対する結合パートナーである。リガンドは、受容体に対する内因性のリガンドであるか、またはその代わりに、薬剤、薬剤候補、もしくは薬理学的手段のような受容体に対する合成リガンドであり得る。 As used herein, a "ligand" is a specific receptor or binding partner to a family of receptors. The ligand can be an endogenous ligand for the receptor or, instead, a synthetic ligand for the receptor such as a drug, drug candidate, or pharmacological means.
　本明細書において「抗体」は、広義には、ポリクローナル抗体、モノクローナル抗体、多重特異性抗体、キメラ抗体、および抗イディオタイプ抗体、ならびにそれらの機能性フラグメント（例えば、Ｆ（ａｂ’）２、およびＦａｂフラグメント）、ならびにその他の組換えにより生産された結合体または機能的等価物（例えば、キメラ抗体、ヒト化抗体、多機能抗体、二重特異性またはオリゴ特異性（oligospecific）抗体、単鎖抗体、一本鎖抗体（scFV）、ダイアボディー（diabody）、sc（Fv）2（single　chain　（Fv）2）、scFv－Fc）を含む。さらにこのような抗体を、酵素、例えばアルカリホスファターゼ、西洋ワサビペルオキシダーゼ、αガラクトシダーゼなど、に共有結合させまたは組換えにより融合させてよい。さらにこのような抗体を、酵素、例えばアルカリホスファターゼ、西洋ワサビペルオキシダーゼ、αガラクトシダーゼなど、に共有結合させまたは組換えにより融合させてよい。狭義に使用する場合は、抗体は、全長抗体（例えば、ポリクローナル抗体、モノクローナル抗体等）を指し、その他は改変体または抗原結合フラグメントと称することがある。本開示で用いられる抗体は、その標的に結合すればよく、その由来、種類、形状などは問われない。具体的には、非ヒト動物の抗体（例えば、マウス抗体、ラット抗体、ラクダ抗体）、ヒト抗体、キメラ抗体、ヒト化抗体などの公知の抗体をもとに製造することができる。本開示においては、一本鎖抗体が使用される。抗体の標的への結合は識別的なあるいは特異的な結合であることが好ましい。抗体の改変体は、抗体と、例えばポリエチレングリコール等の各種分子が結合していてもよい。抗体の改変体は、抗体に公知の手法を用いて化学的な修飾を施すことによって得ることができる。 In the present specification, "antibody" is broadly referred to as a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a chimeric antibody, and an anti-idiotype antibody, and a functional fragment thereof (for example, F (ab') 2 and the like. Fab fragments), as well as conjugates or functional equivalents produced by other recombination (eg, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies). , Single chain antibody (scFV), diabody, sc (Fv) 2 (single chain (Fv) 2 ), scFv-Fc). Further, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase and the like. Further, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase and the like. When used in a narrow sense, an antibody refers to a full-length antibody (eg, polyclonal antibody, monoclonal antibody, etc.), and the others may be referred to as a variant or an antigen-binding fragment. The antibody used in the present disclosure may bind to its target, and its origin, type, shape, etc. are not limited. Specifically, it can be produced based on known antibodies such as non-human animal antibodies (for example, mouse antibody, rat antibody, camel antibody), human antibody, chimeric antibody, and humanized antibody. Single chain antibodies are used in the present disclosure. The binding of the antibody to the target is preferably a discriminative or specific binding. The variant of the antibody may be a combination of the antibody and various molecules such as polyethylene glycol. Variants of the antibody can be obtained by chemically modifying the antibody using known techniques.
　本明細書において「一本鎖抗体」とは、「ｓｃＦｖ（ｓｉｎｇｌｅ　ｃｈａｉｎ　Ｆｖ）」ともいい、抗体の重鎖および軽鎖の可変部領域（ＶＨおよびＶＬ）を適当なリンカーペプチドで連結させたものに相当する。このようなコンストラクトを遺伝子レベルで構築し、タンパク質発現用ベクターを用いて大腸菌に導入することで一本鎖抗体タンパク質を発現させることができる。 As used herein, the term "single chain antibody" is also referred to as "scFv (single chain Fv)", and the variable region regions ( VH and VL ) of the heavy and light chains of the antibody are linked with appropriate linker peptides. Corresponds to the one. A single-chain antibody protein can be expressed by constructing such a construct at the gene level and introducing it into Escherichia coli using a protein expression vector.
　本明細書において、「フラグメント」とは、全長のポリペプチドまたはポリヌクレオチド（長さがｎ）に対して、１～ｎ－１までの配列長を有するポリペプチドまたはポリヌクレオチドをいう。フラグメントの長さは、その目的に応じて、適宜変更することができ、例えば、その長さの下限としては、ポリペプチドの場合、３、４、５、６、７、８、９、１０、１５、２０、２５、３０、４０、５０、およびそれ以上のアミノ酸が挙げられ、ここの具体的に列挙していない整数で表される長さ（例えば、１１など）もまた、下限として適切であり得る。また、ポリヌクレオチドの場合、５、６、７、８、９、１０、１５、２０、２５、３０、４０、５０、７５、１００およびそれ以上のヌクレオチドが挙げられ、ここの具体的に列挙していない整数で表される長さ（例えば、１１など）もまた、下限として適切であり得る。本明細書において、ポリペプチドおよびポリヌクレオチドの長さは、上述のようにそれぞれアミノ酸または核酸の個数で表すことができるが、上述の個数は絶対的なものではなく、同じ機能を有する限り、上限または下限としての上述の個数は、その個数の上下数個（または例えば上下１０％）のものも含むことが意図される。本明細書において有用なフラグメントの長さは、そのフラグメントの基準となる全長タンパク質の機能のうち少なくとも１つの機能が保持されているかどうかによって決定され得る。 As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length from 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). The length of the fragment can be appropriately changed according to its purpose. For example, in the case of a polypeptide, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, and so on. Amino acids such as 15, 20, 25, 30, 40, 50, and above are mentioned, and lengths represented by integers not specifically listed here (eg, 11) are also suitable as lower limits. possible. Further, in the case of polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides are mentioned, and are specifically listed here. A length represented by a non-integer (eg, 11) may also be a suitable lower bound. In the present specification, the lengths of polypeptides and polynucleotides can be expressed by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute and may have an upper limit as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include several above and below the number (or, for example, 10% above and below). The length of a fragment useful herein can be determined by whether at least one of the functions of the full-length protein on which the fragment is based is retained.
　本明細書において遺伝子の「相同性」とは、２以上の遺伝子配列の、互いに対する同一性の程度をいう。従って、ある２つの遺伝子の相同性が高いほど、それらの配列の同一性または類似性は高い。２種類の遺伝子が相同性を有するか否かは、配列の直接の比較、または核酸の場合ストリンジェントな条件下でのハイブリダイゼーション法によって調べられ得る。２つの遺伝子配列を直接比較する場合、その遺伝子配列間でＤＮＡ配列が、代表的には少なくとも５０％同一である場合、好ましくは少なくとも７０％同一である場合、より好ましくは少なくとも８０％、９０％、９５％、９６％、９７％、９８％または９９％同一である場合、それらの遺伝子は相同性を有する。 As used herein, the term "homology" of a gene means the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether or not the two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization under stringent conditions. When two gene sequences are directly compared, the DNA sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%. , 95%, 96%, 97%, 98% or 99%, the genes are homologous.
　アミノ酸は、その一般に公知の３文字記号か、またはＩＵＰＡＣ－ＩＵＢ　Ｂｉｏｃｈｅｍｉｃａｌ　Ｎｏｍｅｎｃｌａｔｕｒｅ　Ｃｏｍｍｉｓｓｉｏｎにより推奨される１文字記号のいずれかにより、本明細書中で言及され得る。ヌクレオチドも同様に、一般に認知された１文字コードにより言及され得る。 Amino acids can be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one-letter code.
　本明細書では、アミノ酸配列および塩基配列の類似性、同一性および相同性の比較は、配列分析用ツールであるＢＬＡＳＴを用いてデフォルトパラメータを用いて算出される。同一性の検索は例えば、ＮＣＢＩのＢＬＡＳＴ　２．９（２０１９．３．１１　発行）を用いて行うことができる。本明細書における同一性の値は通常は上記ＢＬＡＳＴを用い、デフォルトの条件でアラインした際の値をいう。ただし、パラメータの変更により、より高い値が出る場合は、最も高い値を同一性の値とする。複数の領域で同一性が評価される場合はそのうちの最も高い値を同一性の値とする。 In the present specification, comparison of similarity, identity and homology of amino acid sequence and base sequence is calculated using default parameters using BLAST, which is a tool for sequence analysis. The identity search can be performed using, for example, NCBI's BLAST 2.9 (issued on March 11, 2019). The value of identity in the present specification usually refers to the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value.
　本明細書において、「改変体」とは、もとのポリペプチドまたはポリヌクレオチドなどの物質に対して、一部が変更されているものをいう。そのような改変体としては、置換改変体、付加改変体、欠失改変体、短縮（ｔｒｕｎｃａｔｅｄ）改変体、対立遺伝子変異体などが挙げられる。対立遺伝子（ａｌｌｅｌｅ）とは、同一遺伝子座に属し、互いに区別される遺伝的改変体のことをいう。従って、「対立遺伝子変異体」とは、ある遺伝子に対して、対立遺伝子の関係にある改変体をいう。「種相同体またはホモログ（ｈｏｍｏｌｏｇ）」とは、ある種の中で、ある遺伝子とアミノ酸レベルまたはヌクレオチドレベルで、相同性（好ましくは、６０％以上の相同性、より好ましくは、８０％以上、８５％以上、９０％以上、９５％以上の相同性）を有するものをいう。そのような種相同体を取得する方法は、本明細書の記載から明らかである。「オルソログ（ｏｒｔｈｏｌｏｇ）」とは、オルソロガス遺伝子（ｏｒｔｈｏｌｏｇｏｕｓ　ｇｅｎｅ）ともいい、二つの遺伝子がある共通祖先からの種分化に由来する遺伝子をいう。例えば、多重遺伝子構造をもつヘモグロビン遺伝子ファミリーを例にとると、ヒトとマウスのαヘモグロビン遺伝子はオルソログであるが，ヒトのαヘモグロビン遺伝子とβヘモグロビン遺伝子はパラログ（遺伝子重複で生じた遺伝子）である。オルソログは、分子系統樹の推定に有用であることから、オルソログもまた、本開示において有用であり得る。 As used herein, the term "modified" means a substance that has been partially modified from the original polypeptide or substance such as a polynucleotide. Such variants include substitution variants, addition variants, deletion variants, truncated variants, allelic variants and the like. An allele is a genetic variant that belongs to the same locus and is distinguished from each other. Therefore, the "allele variant" refers to a variant having an allele relationship with a certain gene. A "species homolog or homolog" is, within a species, homologous (preferably 60% or more, more preferably 80% or more,) at the amino acid or nucleotide level with a gene. Those having 85% or more, 90% or more, 95% or more homology). The method for obtaining such species homologues is clear from the description herein. "Ortholog" is also called an orthologous gene, and refers to a gene derived from speciation from a common ancestor having two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse α-hemoglobin genes are orthologs, while the human α-hemoglobin and β-hemoglobin genes are paralogs (genes generated by gene duplication). .. Since orthologs are useful for estimating molecular phylogenetic trees, orthologs may also be useful in the present disclosure.
　本明細書において「保存的（に改変された）改変体」は、アミノ酸配列および核酸配列の両方に適用される。特定の核酸配列に関して、保存的に改変された改変体とは、同一のまたは本質的に同一のアミノ酸配列をコードする核酸をいい、核酸がアミノ酸配列をコードしない場合には、本質的に同一な配列をいう。このような塩基配列の改変法としては、制限酵素などによる切断、ＤＮＡポリメラーゼ、Ｋｌｅｎｏｗフラグメント、ＤＮＡリガーゼなどによる処理等による連結等の処理、合成オリゴヌクレオチドなどを用いた部位特異的塩基置換法（特定部位指向突然変異法；ＭａｒｋＺｏｌｌｅｒａｎｄ　Ｍｉｃｈａｅｌ　Ｓｍｉｔｈ，Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，１００，４６８－５００（１９８３））が挙げられるが、この他にも通常分子生物学の分野で用いられる方法によって改変を行うこともできる。遺伝コードの縮重のため、多数の機能的に同一な核酸が任意の所定のタンパク質をコードする。例えば、コドンＧＣＡ、ＧＣＣ、ＧＣＧ、およびＧＣＵはすべて、アミノ酸アラニンをコードする。したがって、アラニンがコドンにより特定される全ての位置で、そのコドンは、コードされたポリペプチドを変更することなく、記載された対応するコドンの任意のものに変更され得る。このような核酸の変動は、保存的に改変された変異の１つの種である「サイレント改変（変異）」である。ポリペプチドをコードする本明細書中のすべての核酸配列はまた、その核酸の可能なすべてのサイレント変異を記載する。当該分野において、核酸中の各コドン（通常メチオニンのための唯一のコドンであるＡＵＧ、および通常トリプトファンのための唯一のコドンであるＴＧＧを除く）が、機能的に同一な分子を産生するために改変され得ることが理解される。したがって、ポリペプチドをコードする核酸の各サイレント変異は、記載された各配列において暗黙に含まれる。好ましくは、そのような改変は、ポリペプチドの高次構造に多大な影響を与えるアミノ酸であるシステインの置換を回避するようになされ得る。 As used herein, the term "conservative (modified) variant" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, a conservatively modified variant is a nucleic acid that encodes the same or essentially the same amino acid sequence, and if the nucleic acid does not encode an amino acid sequence, it is essentially the same. Refers to an array. Such base sequence modification methods include cleavage with restriction enzymes, ligation by treatment with DNA polymerase, Klenow fragment, DNA ligase, etc., and site-specific base substitution method using synthetic oligonucleotides (specification). Site-directed mutation method; MarkZollerand Michael Smith, Methods in Enzymology, 100, 468-500 (1983)), but other methods usually used in the field of molecular biology can also be used for modification. Due to the degeneracy of the genetic code, multiple functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is identified by a codon, that codon can be changed to any of the corresponding corresponding codons described without changing the encoded polypeptide. Such nucleic acid variation is a "silent modification (mutation)", which is a type of conservatively modified mutation. All nucleic acid sequences herein that encode a polypeptide also describe all possible silent mutations of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) to produce a functionally identical molecule. It is understood that it can be modified. Therefore, each silent mutation in the nucleic acid encoding the polypeptide is implicitly included in each of the sequences described. Preferably, such modifications can be made to avoid substitutions of the amino acid cysteine, which has a profound effect on the higher order structure of the polypeptide.
　あるアミノ酸は、相互作用結合能力の明らかな低下または消失なしに、例えば、リガンド分子の結合部位のようなタンパク質構造において他のアミノ酸に置換され得る。あるタンパク質の生物学的機能を規定するのは、タンパク質の相互作用能力および性質である。従って、特定のアミノ酸の置換がアミノ酸配列において、またはそのＤＮＡコード配列のレベルにおいて行われ得、置換後もなお、もとの性質を維持するタンパク質が生じ得る。従って、生物学的有用性の明らかな損失なしに、種々の改変が、本明細書において開示されたペプチドまたはこのペプチドをコードする対応するＤＮＡにおいて行われ得る。 One amino acid can be replaced by another amino acid in a protein structure, such as the binding site of a ligand molecule, without a significant reduction or loss of the ability to interact. It is the ability and nature of a protein to interact that defines the biological function of a protein. Thus, substitutions of a particular amino acid can be made at the amino acid sequence or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after substitution. Thus, various modifications can be made in the peptides disclosed herein or in the corresponding DNA encoding the peptides, without any apparent loss of biological usefulness.
　このような核酸は、周知のＰＣＲ法により得ることができ、化学的に合成することもできる。これらの方法に、例えば、部位特異的変位誘発法、ハイブリダイゼーション法などを組み合わせてもよい。 Such nucleic acids can be obtained by a well-known PCR method and can also be chemically synthesized. For example, a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.
　上記のような改変を設計する際に、アミノ酸の疎水性指数が考慮され得る。タンパク質における相互作用的な生物学的機能を与える際の疎水性アミノ酸指数の重要性は、一般に当該分野で認められている（Ｋｙｔｅ．ＪおよびＤｏｏｌｉｔｔｌｅ，Ｒ．Ｆ．Ｊ．Ｍｏｌ．Ｂｉｏｌ．１５７（１）：１０５－１３２，１９８２）。アミノ酸の疎水的性質は、生成したタンパク質の二次構造に寄与し、次いでそのタンパク質と他の分子（例えば、酵素、基質、受容体、ＤＮＡ、抗体、抗原など）との相互作用を規定する。各アミノ酸は、それらの疎水性および電荷の性質に基づく疎水性指数を割り当てられる。それらは：イソロイシン（＋４．５）；バリン（＋４．２）；ロイシン（＋３．８）；フェニルアラニン（＋２．８）；システイン／シスチン（＋２．５）；メチオニン（＋１．９）；アラニン（＋１．８）；グリシン（－０．４）；スレオニン（－０．７）；セリン（－０．８）；トリプトファン（－０．９）；チロシン（－１．３）；プロリン（－１．６）；ヒスチジン（－３．２）；グルタミン酸（－３．５）；グルタミン（－３．５）；アスパラギン酸（－３．５）；アスパラギン（－３．５）；リジン（－３．９）；およびアルギニン（－４．５））である。 Amino acid hydrophobicity index can be taken into account when designing modifications as described above. The importance of the hydrophobic amino acid index in imparting interactive biological functions in proteins is generally recognized in the art (Kyte.J and Dollittle, LFJ. Mol.Biol.157 (Kyte.J and Doolittle, LFJ. Mol.Biol.157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced, which in turn defines the interaction of that protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1) .8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6) ); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Aspartic acid (-3.5); Leucine (-3.9) And arginine (-4.5)).
　あるアミノ酸を、同様の疎水性指数を有する他のアミノ酸により置換して、そして依然として同様の生物学的機能を有するタンパク質（例えば、リガンド結合能において等価なタンパク質）を生じさせ得ることが当該分野で周知である。このようなアミノ酸置換において、疎水性指数が±２以内であることが好ましく、±１以内であることがより好ましく、および±０．５以内であることがさらにより好ましい。疎水性に基づくこのようなアミノ酸の置換は効率的であることが当該分野において理解される。米国特許第４，５５４，１０１号に記載されるように、以下の親水性指数がアミノ酸残基に割り当てられている：アルギニン（＋３．０）；リジン（＋３．０）；アスパラギン酸（＋３．０±１）；グルタミン酸（＋３．０±１）；セリン（＋０．３）；アスパラギン（＋０．２）；グルタミン（＋０．２）；グリシン（０）；スレオニン（－０．４）；プロリン（－０．５±１）；アラニン（－０．５）；ヒスチジン（－０．５）；システイン（－１．０）；メチオニン（－１．３）；バリン（－１．５）；ロイシン（－１．８）；イソロイシン（－１．８）；チロシン（－２．３）；フェニルアラニン（－２．５）；およびトリプトファン（－３．４）。アミノ酸が同様の親水性指数を有しかつ依然として生物学的等価体を与え得る別のものに置換され得ることが理解される。このようなアミノ酸置換において、親水性指数が±２以内であることが好ましく、±１以内であることがより好ましく、および±０．５以内であることがさらにより好ましい。 It is possible in the art to replace one amino acid with another amino acid having a similar hydrophobicity index and to produce a protein that still has similar biological functions (eg, a protein equivalent in ligand binding ability). It is well known. In such amino acid substitutions, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such substitutions of amino acids based on hydrophobicity are efficient. The following hydrophilicity indices are assigned to amino acid residues as described in US Pat. No. 4,554,101: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); aspartic acid (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline ( -0.5 ± 1); alanin (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.5) -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be replaced by another that has a similar hydrophilicity index and can still give a bioisostere. In such amino acid substitutions, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.
　本開示において、「保存的置換」とは、アミノ酸置換において、元のアミノ酸と置換されるアミノ酸との親水性指数または／および疎水性指数が上記のように類似している置換をいう。保存的置換の例は、当業者に周知であり、例えば、次の各グループ内での置換：アルギニンおよびリジン；グルタミン酸およびアスパラギン酸；セリンおよびスレオニン；グルタミンおよびアスパラギン；ならびにバリン、ロイシン、およびイソロイシン、などが挙げられるがこれらに限定されない。 In the present disclosure, "conservative substitution" refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced is similar to that of the original amino acid in the amino acid substitution as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and aspartic acid; and valine, leucine, and isoleucine, However, it is not limited to these.
　本明細書中において、機能的に等価なポリペプチドを作製するために、アミノ酸の置換のほかに、アミノ酸の付加、欠失、または修飾もまた行うことができる。アミノ酸の置換とは、もとのペプチドを１つ以上、例えば、１～１０個、好ましくは１～５個、より好ましくは１～３個のアミノ酸で置換することをいう。アミノ酸の付加とは、もとのペプチド鎖に１つ以上、例えば、１～１０個、好ましくは１～５個、より好ましくは１～３個のアミノ酸を付加することをいう。アミノ酸の欠失とは、もとのペプチドから１つ以上、例えば、１～１０個、好ましくは１～５個、より好ましくは１～３個のアミノ酸を欠失させることをいう。アミノ酸修飾は、アミド化、カルボキシル化、硫酸化、ハロゲン化、アルキル化、リン酸化、水酸化、アシル化（例えば、アセチル化）などを含むが、これらに限定されない。置換、または付加されるアミノ酸は、天然のアミノ酸であってもよく、非天然のアミノ酸、またはアミノ酸アナログでもよい。天然のアミノ酸が好ましい。 In addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made herein to make functionally equivalent polypeptides. Amino acid substitution refers to substituting one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids of the original peptide. The addition of amino acids means the addition of one or more amino acids, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids to the original peptide chain. Amino acid deletion refers to the deletion of one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, phosphorylation, hydroxylation, acylation (eg, acetylation) and the like. The amino acid substituted or added may be a natural amino acid, an unnatural amino acid, or an amino acid analog. Natural amino acids are preferred.
　本明細書において、ポリペプチドまたはポリヌクレオチドの「置換、付加および／または欠失」とは、もとのポリペプチドまたはポリヌクレオチドに対して、それぞれアミノ酸もしくはその代替物、またはヌクレオチドもしくはその代替物が、置き換わること、付け加わること、または取り除かれることをいう。このような置換、付加および／または欠失の技術は、当該分野において周知であり、そのような技術の例としては、部位特異的変異誘発技術などが挙げられる。基準となる核酸分子またはポリペプチドにおけるこれらの変化は、目的とする機能（例えば、ＲＡＧＥの認識能など）が保持される限り、この核酸分子の５’末端もしくは３’末端で生じ得るか、またはこのポリペプチドを示すアミノ酸配列のアミノ末端部位もしくはカルボキシ末端部位で生じ得るか、またはそれらの末端部位の間のどこにでも生じ得、基準配列中の残基間で個々に散在する。置換、付加または欠失は、１つ以上であれば任意の数でよく、そのような数は、その置換、付加または欠失を有する改変体において目的とする機能が保持される限り、多くすることができる。例えば、そのような数は、１または数個であり得、そして好ましくは、全体の長さの２０％以内、１５％以内、１０％以内、５％以内、または１５０個以下、１００個以下、５０個以下、２５個以下などであり得る。 As used herein, the term "substitution, addition and / or deletion" of a polypeptide or polynucleotide means that the original polypeptide or polynucleotide is an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively. , To be replaced, to be added, or to be removed. Techniques for such substitutions, additions and / or deletions are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. These changes in the reference nucleic acid molecule or polypeptide can occur at the 5'end or 3'end of the nucleic acid molecule, as long as the desired function (eg, RAGE recognition) is retained. It can occur at the amino or carboxy-terminal sites of the amino acid sequences that represent this polypeptide, or can occur anywhere between those terminal sites and are individually interspersed among the residues in the reference sequence. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such a number may be increased as long as the desired function is retained in the variant having the substitution, addition or deletion. be able to. For example, such numbers can be one or several, and preferably within 20%, within 15%, within 10%, within 5%, or below 150, below 100, of the total length. It can be 50 or less, 25 or less, and so on.
　本明細書において使用される場合、用語「タグ配列」とは、受容体－リガンドのような特異的認識機構により分子を選別するための物質、より具体的には、特定の物質を結合するための結合パートナーの役割を果たす物質（例えば、ビオチン－アビジン、ビオチン－ストレプトアビジンのような関係を有する）をいう。よって、例えば、タグ配列が結合した特定の物質は、タグ配列の結合パートナーを結合させた基材を接触させることで、この特定の物質を選別することができる。このようなタグ配列は、当該分野で周知である。代表的なタグ配列としては、ｍｙｃタグ、Ｈｉｓタグ、ＨＡ、Ａｖｉタグなどが挙げられるが、これらに限定されない。 As used herein, the term "tag sequence" is used to bind a substance for selecting a molecule by a specific recognition mechanism such as a receptor-ligand, more specifically, a specific substance. A substance that acts as a binding partner of (for example, having a relationship such as biotin-avidin and biotin-streptavidin). Therefore, for example, a specific substance to which the tag sequence is bound can be selected by contacting the base material to which the binding partner of the tag sequence is bound. Such tag sequences are well known in the art. Typical tag sequences include, but are not limited to, myc tags, His tags, HA, Avi tags, and the like.
　本明細書において「タンパク質」、「ポリペプチド」、「オリゴペプチド」および「ペプチド」は、本明細書において同じ意味で使用され、任意の長さのアミノ酸のポリマーをいう。このポリマーは、直鎖であっても分岐していてもよく、環状であってもよい。アミノ酸は、天然のものであっても非天然のものであってもよく、改変されたアミノ酸であってもよい。この用語はまた、複数のポリペプチド鎖の複合体へとアセンブルされたものを包含し得る。この用語はまた、天然または人工的に改変されたアミノ酸ポリマーも包含する。そのような改変としては、例えば、ジスルフィド結合形成、グリコシル化、脂質化、アセチル化、リン酸化または任意の他の操作もしくは改変（例えば、標識成分との結合体化）。この定義にはまた、例えば、アミノ酸の１または２以上のアナログを含むポリペプチド（例えば、非天然アミノ酸などを含む）、ペプチド様化合物（例えば、ペプトイド）および当該分野において公知の他の改変が包含される。 In the present specification, "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably in the present specification and refer to a polymer of amino acids of any length. The polymer may be linear, branched or cyclic. The amino acid may be natural or non-natural, or may be a modified amino acid. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). The definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids), peptide-like compounds (eg, peptoids) and other modifications known in the art. Will be done.
　本明細書において、「アミノ酸」は、本開示の目的を満たす限り、天然のものでも非天然のものでもよい。本明細書において「アミノ酸誘導体」または「アミノ酸アナログ」とは、天然に存在するアミノ酸とは異なるがもとのアミノ酸と同様の機能を有するものをいう。そのようなアミノ酸誘導体およびアミノ酸アナログは、当該分野において周知である。本明細書では、アミノ酸誘導体およびアミノ酸アナログは、アミノ酸と同じ生物学的機能を提供し得る限り代替として使用され得ることが理解される。本明細書において「天然のアミノ酸」とは、天然のアミノ酸のＬ－異性体を意味する。天然のアミノ酸は、グリシン、アラニン、バリン、ロイシン、イソロイシン、セリン、メチオニン、トレオニン、フェニルアラニン、チロシン、トリプトファン、システイン、プロリン、ヒスチジン、アスパラギン酸、アスパラギン、グルタミン酸、グルタミン、γ－カルボキシグルタミン酸、アルギニン、オルニチン、およびリジンである。特に示されない限り、本明細書でいう全てのアミノ酸はＬ体であるが、Ｄ体のアミノ酸を用いた形態もまた本開示の範囲内にある。本明細書において「非天然アミノ酸」とは、タンパク質中で通常は天然に見出されないアミノ酸を意味する。非天然アミノ酸の例として、ノルロイシン、パラ－ニトロフェニルアラニン、ホモフェニルアラニン、パラ－フルオロフェニルアラニン、３－アミノ－２－ベンジルプロピオン酸、ホモアルギニンのＤ体またはＬ体およびＤ－フェニルアラニンが挙げられる。本明細書において「アミノ酸アナログ」とは、アミノ酸ではないが、アミノ酸の物性および／または機能に類似する分子をいう。アミノ酸アナログとしては、例えば、エチオニン、カナバニン、２－メチルグルタミンなどが挙げられる。アミノ酸模倣物とは、アミノ酸の一般的な化学構造とは異なる構造を有するが、天然に存在するアミノ酸と同様な様式で機能する化合物をいう。 In the present specification, the "amino acid" may be natural or non-natural as long as it satisfies the object of the present disclosure. As used herein, the term "amino acid derivative" or "amino acid analog" refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such amino acid derivatives and amino acid analogs are well known in the art. It is understood herein that amino acid derivatives and amino acid analogs can be used as alternatives as long as they can provide the same biological functions as amino acids. As used herein, the term "natural amino acid" means the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, aspartic acid, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornitine. , And lysine. Unless otherwise specified, all amino acids referred to herein are L-form, but forms using D-form amino acids are also within the scope of the present disclosure. As used herein, the term "unnatural amino acid" means an amino acid that is not normally found in proteins. Examples of unnatural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, D- or L-form of homoarginine and D-phenylalanine. As used herein, the term "amino acid analog" refers to a molecule that is not an amino acid but is similar in physical properties and / or function to an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics are compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.
　アミノ酸は、その一般に公知の３文字記号か、またはＩＵＰＡＣ－ＩＵＢ　Ｂｉｏｃｈｅｍｉｃａｌ　Ｎｏｍｅｎｃｌａｔｕｒｅ　Ｃｏｍｍｉｓｓｉｏｎにより推奨される１文字記号のいずれかにより、本明細書中で言及され得る。ヌクレオチドも同様に、一般に認知された１文字コードにより言及され得る。 Amino acids can be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one-letter code.
　本明細書において「ポリヌクレオチド」、「オリゴヌクレオチド」および「核酸」は、本明細書において同じ意味で使用され、任意の長さのヌクレオチドのポリマーをいう。この用語はまた、「オリゴヌクレオチド誘導体」または「ポリヌクレオチド誘導体」を含む。「オリゴヌクレオチド誘導体」または「ポリヌクレオチド誘導体」とは、ヌクレオチドの誘導体を含むか、またはヌクレオチド間の結合が通常とは異なるオリゴヌクレオチドまたはポリヌクレオチドをいい、互換的に使用される。そのようなオリゴヌクレオチドとして具体的には、例えば、２’－Ｏ－メチル－リボヌクレオチド、オリゴヌクレオチド中のリン酸ジエステル結合がホスホロチオエート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のリン酸ジエステル結合がＮ３’－Ｐ５’ホスホロアミデート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のリボースとリン酸ジエステル結合とがペプチド核酸結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のウラシルがＣ－５プロピニルウラシルで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のウラシルがＣ－５チアゾールウラシルで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のシトシンがＣ－５プロピニルシトシンで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のシトシンがフェノキサジン修飾シトシン（ｐｈｅｎｏｘａｚｉｎｅ－ｍｏｄｉｆｉｅｄ　ｃｙｔｏｓｉｎｅ）で置換されたオリゴヌクレオチド誘導体、ＤＮＡ中のリボースが２’－Ｏ－プロピルリボースで置換されたオリゴヌクレオチド誘導体およびオリゴヌクレオチド中のリボースが２’－メトキシエトキシリボースで置換されたオリゴヌクレオチド誘導体などが例示される。他にそうではないと示されなければ、特定の核酸配列はまた、明示的に示された配列と同様に、その保存的に改変された改変体（例えば、縮重コドン置換体）および相補配列を包含することが企図される。具体的には、縮重コドン置換体は、１またはそれ以上の選択された（または、すべての）コドンの３番目の位置が混合塩基および／またはデオキシイノシン残基で置換された配列を作成することにより達成され得る（Ｂａｔｚｅｒら、Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓ．１９：５０８１（１９９１）；Ｏｈｔｓｕｋａら、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２６０：２６０５－２６０８（１９８５）；Ｒｏｓｓｏｌｉｎｉら、Ｍｏｌ．Ｃｅｌｌ．Ｐｒｏｂｅｓ　８：９１－９８（１９９４））。 In the present specification, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably in the present specification and refer to a polymer of nucleotides of arbitrary length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". The "oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual bond between nucleotides, and is used interchangeably. Specifically, such an oligonucleotide includes, for example, 2'-O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphate diester bond in an oligonucleotide is converted into a phosphorothioate bond, and a phosphate diester bond in an oligonucleotide. Is an oligonucleotide derivative converted to N3'-P5'phosphoroamidate bond, an oligonucleotide derivative in which ribose and phosphate diester bond in the oligonucleotide are converted into peptide nucleic acid bond, and uracil in the oligonucleotide is C- 5 Oligonucleotide derivatives substituted with propynyl uracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced with C-5 thiazole uracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced with C-5 propynyl citosine, oligonucleotides Oligonucleotide derivatives in which cytosine in nucleotides is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is replaced with 2'-O-propylribose, and ribose in oligonucleotides are 2 Examples thereof include oligonucleotide derivatives substituted with'-methoxyethoxyribose. Unless otherwise indicated, certain nucleic acid sequences are also conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as are the explicitly indicated sequences. Is intended to be included. Specifically, the degenerate codon substituent creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91- 98 (1994)).
　本明細書において「ヌクレオチド」は、天然のものでも非天然のものでもよい。「ヌクレオチド誘導体」または「ヌクレオチドアナログ」とは、天然に存在するヌクレオチドとは異なるがもとのヌクレオチドと同様の機能を有するものをいう。そのようなヌクレオチド誘導体およびヌクレオチドアナログは、当該分野において周知である。そのようなヌクレオチド誘導体およびヌクレオチドアナログの例としては、ホスホロチオエート、ホスホルアミデート、メチルホスホネート、キラルメチルホスホネート、２’－Ｏ－メチルリボヌクレオチド、ペプチド型核酸（ＰＮＡ）が含まれるが、これらに限定されない。 In the present specification, the "nucleotide" may be natural or non-natural. A "nucleotide derivative" or "nucleotide analog" is one that is different from the naturally occurring nucleotide but has the same function as the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, peptide-type nucleic acids (PNAs). Not done.
　本明細書において「核酸」はまた、遺伝子、ｃＤＮＡ、ｍＲＮＡ、オリゴヌクレオチド、およびポリヌクレオチドと互換可能に使用される。特定の核酸配列はまた、「スプライス改変体」を包含する。同様に、核酸によりコードされた特定のタンパク質は、その核酸のスプライス改変体によりコードされる任意のタンパク質を暗黙に包含する。その名が示唆するように「スプライス改変体」は、遺伝子のオルタナティブスプライシングの産物である。転写後、最初の核酸転写物は、異なる（別の）核酸スプライス産物が異なるポリペプチドをコードするようにスプライスされ得る。スプライス改変体の産生機構は変化するが、エキソンのオルタナティブスプライシングを含む。読み過し転写により同じ核酸に由来する別のポリペプチドもまた、この定義に包含される。スプライシング反応の任意の産物（組換え形態のスプライス産物を含む）がこの定義に含まれる。 As used herein, "nucleic acid" is also used interchangeably with genes, cDNAs, mRNAs, oligonucleotides, and polynucleotides. Specific nucleic acid sequences also include "splice variants". Similarly, a particular protein encoded by a nucleic acid implicitly includes any protein encoded by a splice variant of that nucleic acid. As the name suggests, "splice variants" are the product of alternative splicing of genes. After transcription, the first nucleic acid transcript can be spliced so that different (different) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Another polypeptide derived from the same nucleic acid by over-read transcription is also included in this definition. Any product of the splicing reaction, including recombinant splice products, is included in this definition.
　本明細書において「遺伝子」とは、遺伝形質を規定する因子をいう。通常染色体上に一定の順序に配列している。タンパク質の一次構造を規定する遺伝子を構造遺伝子といい、その発現を左右する遺伝子を調節遺伝子という。本明細書では、「遺伝子」は、「ポリヌクレオチド」、「オリゴヌクレオチド」および「核酸」ならびに／あるいは「タンパク質」「ポリペプチド」、「オリゴペプチド」および「ペプチド」をさすことがある。 As used herein, the term "gene" refers to a factor that defines a genetic trait. It is usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that influences its expression is called a regulatory gene. As used herein, "gene" may refer to "polynucleotide", "oligonucleotide" and "nucleic acid" and / or "protein" "polypeptide", "oligopeptide" and "peptide".
　本明細書において、「リン酸緩衝化生理食塩水（ＰＢＳ）とは、ＮａＣｌ、ＫＣｌ、Ｎａ２ＨＰＯ４、およびＫＨ２ＰＯ４を含むｐＨ７～８の水溶液である。各成分の濃度およびｐＨは、用途に応じて好適に調節することができる。本明細書において、「ＰＢＳ（＋）」は、カルシウムイオンおよびマグネシウムイオンを含むことを意味しており、「ＰＢＳ（－）」は、カルシウムイオンおよびマグネシウムイオンを含まないことを意味しているが、本明細書では、明示的に記載しない限り、「ＰＢＳ」は「ＰＢＳ（－）」を意味するものとする。本明細書では、代表的には、Ｄｕｌｂｅｃｃｏ’ｓ　ＰＢＳ（－）を使用することができる。Ｄｕｌｂｅｃｃｏ’ｓ　ＰＢＳ（－）の組成は、ＮａＣｌ　８ｇ，ＫＣｌ　０．２ｇ，Ｎａ２ＨＰＯ４　１．１５ｇ，ＫＨ２ＰＯ４　０．２ｇ／Ｌ、（ｐＨ　７．４）である。 As used herein, "phosphate buffered saline (PBS) is an aqueous solution of pH 7 to 8 containing NaCl, KCl, Na 2 HPO 4 , and KH 2 PO 4. The concentration and pH of each component are In the present specification, "PBS (+)" means containing calcium ion and magnesium ion, and "PBS (-)" means calcium ion. And magnesium ion-free, but herein, "PBS" shall mean "PBS (-)" unless explicitly stated. In the present specification, Dulbecco's PBS (−) can be typically used. The composition of Dulbecco's PBS (−) is NaCl 8 g, KCl 0.2 g, Na 2 HPO 4 1.15 g, KH 2 PO 4 0.2 g / L, (pH 7.4).
　本明細書において使用される場合、用語「受容体」とは、１個以上のリガンドと可逆的、かつ特異的に複合体化する１個以上の結合ドメインを備える生物学的な構造であって、ここで、この複合体化は生物学的な構造を有する。受容体は、完全に細胞の外部（細胞外の受容体）、細胞膜の中（しかし、受容体の部分を細胞外部の環境および細胞質ゾルに向けている）、または完全に細胞の中（細胞内の受容体）に存在し得る。これらはまた、細胞と独立的に機能し得る。細胞膜中の受容体は、細胞を、その境界の外部の空間と連絡（例えば、シグナル伝達）させ、そして細胞の内側および外側への分子およびイオンの輸送において機能させることを可能とする。本明細書において使用する場合、受容体は、受容体全長であっても、受容体のフラグメントであってもよい。 As used herein, the term "receptor" is a biological structure comprising one or more binding domains that reversibly and specifically complex with one or more ligands. Here, this complex has a biological structure. Receptors are either completely outside the cell (extracellular receptors), inside the cell membrane (but directing parts of the receptors to the outside environment and cytosol), or completely inside the cell (intracellular). Can be present in the receptor). They can also function independently of the cell. Receptors in the cell membrane allow cells to communicate (eg, signal transduce) with space outside their boundaries and to function in the transport of molecules and ions inside and outside the cell. As used herein, the receptor may be the full length of the receptor or a fragment of the receptor.
　本明細書において、「抗原抗体反応」とは、当該分野で使用される最も広い意味で用いられ、特に、抗原と抗体との間の特異的結合に基づく反応をいう。検出系としてイムノブロット（ウェスタンブロット）形式を用いることによって、試料中の抗原を検出し、そして定量化するための試薬および方法も提供する。 As used herein, the term "antigen-antibody reaction" is used in the broadest sense used in the art, and in particular, refers to a reaction based on a specific binding between an antigen and an antibody. Reagents and methods for detecting and quantifying antigens in a sample by using the immunoblot (Western blot) format as the detection system are also provided.
　試料
　本明細書において、「カイコ」とは、通常の意味のカイコ（蚕）を意味し、チョウ目（鱗翅目）・カイコガ科に属する昆虫の一種であるものとする。正式和名はカイコガ（学名：Ｂｏｍｂｉｘ　ｍｏｒｉ）で、カイコはこの幼虫の名称だが、一般的にはこの種全般をも指す。クワ（桑）を食餌とし、絹を産生して蛹の繭を作る。カイコは家蚕（かさん）とも呼ばれ、野生に生息する昆虫ではない。カイコの祖先は東アジアに生息するクワコ（Ｂｏｍｂｙｘｍａｎｄａｒｉｎａ）であると考えられている。カイコとクワコは学問的には別種とされるが、これらの雑種は生殖能力をもつ。本明細書では、カイコには、クワコが含まれるものとする。本明細書において「カイコと同様の糖鎖を付与する生物」とは、カイコと同様の糖鎖を付加する能力を有する生物をいい、カイコと同様の糖鎖を付加する酵素をコードする遺伝子等でトランスジェニック生物等を含みうる。 Sample In the present specification, "silk moth" means silk moth (silkmoth) in the usual meaning, and is considered to be a kind of insect belonging to the order Lepidoptera (Lepidoptera) and Bombyx mori. The official Japanese name is the silk moth (scientific name: Bombyx mori), which is the name of this larva, but generally also refers to this species in general. It feeds on mulberry and produces silk to make pupal cocoons. The silk moth is also called a domestic silk moth and is not an insect that lives in the wild. The ancestor of the silk moth is thought to be the Bombyx mandarina, which inhabits East Asia. Spirogyx and Bombyx mandarin are academically distinct species, but these hybrids are fertile. In the present specification, the silk moth includes the mulberry. As used herein, the term "organism that imparts sugar chains similar to silk moth" refers to an organism that has the ability to add sugar chains similar to silk moth, such as a gene encoding an enzyme that adds sugar chains similar to silk moth. Can include transgenic organisms and the like.
　本明細書において、「絹糸腺」とは、熟蚕の体内に存在する左右１対の器官であり、クワの葉から摂取した多量のタンパク質（アミノ酸）を２種類の絹タンパク質（フィブロイン、セリシン）に変える器官を意味する。絹糸腺は左右一対となっており、マユ糸の原料となる液状絹を分泌する。絹糸腺は、後部絹糸腺、中部絹糸腺、前部絹糸腺の３つの部分に分けられる。本開示では、いずれの絹糸腺を用いても合成可能であるが、合成後の取り扱いを考慮し、後部絹糸腺、中部絹糸腺が通常使用され、好ましくは中部絹糸腺が使用されるが、それに限定されるものではない。また、全身に発現させて全身から回収することも可能であるし、マユを形成させた後にマユから回収することも可能である。 In the present specification, the "silk gland" is a pair of left and right organs existing in the body of a mature silk moth, and a large amount of protein (amino acid) ingested from mulberry leaves is combined with two types of silk proteins (fibroin and sericin). Means an organ that changes to. The silk glands are paired on the left and right, and secrete liquid silk, which is the raw material for eyebrows. The silk gland is divided into three parts: a posterior silk gland, a middle silk gland, and an anterior silk gland. In the present disclosure, any silk gland can be used for synthesis, but in consideration of post-synthesis handling, the posterior silk gland and the middle silk gland are usually used, and the middle silk gland is preferably used. It is not limited. In addition, it can be expressed in the whole body and recovered from the whole body, or it can be recovered from the eyebrows after forming the eyebrows.
　後部絹糸腺とは、最後部にある細長い部分で、伸ばすと約２０ｃｍになる。ここでは後にマユ糸の中心となるフィブロインタンパク質を合成する。 The posterior silk gland is an elongated part at the rearmost part, which becomes about 20 cm when extended. Here, the fibroin protein, which is the center of the eyebrows, is synthesized later.
　中部絹糸腺とは、中央部分にあるＳ字に曲がった太い部分で、伸ばすと約６ｃｍになる。後部絹糸腺から送られてきたフィブロインタンパク質を濃縮して蓄え、繊維にしやすい形に整える。またもうひとつの絹タンパク質であるセリシンも分泌する。マユ糸を吐きだすとき、フィブロインタンパク質をまとめる接着剤の役割をする。 The central silk gland is a thick part that is bent in an S shape in the central part, and when extended, it becomes about 6 cm. The fibroin protein sent from the posterior silk gland is concentrated and stored, and shaped into fibers. It also secretes another silk protein, sericin. When spitting out eyebrows, it acts as an adhesive that holds fibroin proteins together.
　前部絹糸腺とは、長さは約４ｃｍの吐糸口につながる細い管で、先に行くほど細くなる。液状のフィブロインタンパク質の分子が引き伸ばされて一定方向に揃えられ、互いに集合することでさらに水分が除かれる。管の先端でもう一対の管と一本に合流し、吐糸口から吐きだされて一本のマユ糸になる。 The anterior silk gland is a thin tube that connects to the spout, which is about 4 cm in length, and becomes thinner toward the end. Molecules of liquid fibroin protein are stretched and aligned in a certain direction and aggregate with each other to further remove water. At the tip of the tube, it merges with another pair of tubes and is spit out from the spout to become a single eyebrows.
　カイコは５齢の終わり頃にクワを食べるのをやめる（熟蚕）。熟蚕の体の中は、マユ糸の原料となる水飴のような液（液状絹）をため込んだ一対の器官（絹糸腺）でいっぱいになっている。絹糸腺は、細い吐糸管をつうじてカイコの口元にある吐糸口につながっている。液状絹は細い吐糸管を通ることで引き伸ばされて固まり、マユ糸となる。さらには、幼虫が吐糸管から吐きだした糸を近くのものに貼りつけ、頭と胸を８の字状に動かし、引っぱるという一連の運動で、マユ糸は次々と絹糸腺から引きだされるのである。 Silk moth stops eating mulberry at the end of the 5th instar (mature silk moth). The body of the tussah is filled with a pair of organs (silk glands) that store a liquid (liquid silk) like starch syrup, which is the raw material for eyebrows. The silk gland is connected to the spout at the mouth of the silk moth through a thin spit tube. Liquid silk is stretched and hardened by passing through a thin spit tube to become eyebrows. Furthermore, the eyebrows are pulled out from the silk glands one after another by a series of movements in which the thread spit out from the spit tube by the larva is attached to a nearby object, the head and chest are moved in a figure eight shape, and the thread is pulled. It is.
　本明細書において「カイコ型糖鎖」とは、カイコで生産される糖タンパク質において特有の糖鎖構造をいい、代表的にはトリマンノシルコア（自体）、オリゴマンノース型糖鎖および複合型糖鎖、あるいはそのハイブリッド型がある。本開示では、中部絹糸腺を用いてカイコ型糖タンパク質を生産していることから、特に断らない限り、「カイコ型糖鎖」とは、この中部絹糸腺で生産される特有の糖鎖型をいう。このようなカイコ型糖鎖としては、例えば、アスパラギン（Ａｓｎ）に結合したＮアセチルグルコサミン（ＧｌｃＮＡｃ）が２個結合した後、マンノース（Ｍａｎ）が３分子結合したもの（トリマンノシルコアといい、下記（１）式で示される）をコアとして、そこから分岐した構造をとり、さらに種々の糖鎖が結合している。 As used herein, the term "silk moth-type sugar chain" refers to a sugar chain structure peculiar to glycoproteins produced in silk moth, and is typically trimannosyl core (itself), oligomannose-type sugar chain, and complex-type sugar chain. , Or its hybrid type. In the present disclosure, since the silk moth-type glycoprotein is produced using the central silk gland, unless otherwise specified, the "silk moth-type sugar chain" refers to the specific sugar chain type produced by the central silk gland. Say. Examples of such a silkworm-type sugar chain include two N-acetylglucosamines (GlcNAc) bound to asparagine (Asn) and then three molecular bindings of mannose (Man) (referred to as trimannosyl core, described below. It has a structure branched from the core (represented by Eq. (1)), and various sugar chains are further bound to it.
　本明細書において「対応する」アミノ酸または核酸とは、あるポリペプチド分子またはポリヌクレオチド分子において、比較の基準となるポリペプチドまたはポリヌクレオチドにおける所定のアミノ酸またはヌクレオチドと同様の作用を有するか、または有することが予測されるアミノ酸またはヌクレオチドをいい、特に酵素分子にあっては、活性部位中の同様の位置に存在し触媒活性に同様の寄与をするアミノ酸をいう。例えば、アンチセンス分子であれば、そのアンチセンス分子の特定の部分に対応するオルソログにおける同様の部分であり得る。対応するアミノ酸は、例えば、システイン化、グルタチオン化、Ｓ－Ｓ結合形成、酸化（例えば、メチオニン側鎖の酸化）、ホルミル化、アセチル化、リン酸化、糖鎖付加、ミリスチル化などがされる特定のアミノ酸であり得る。あるいは、対応するアミノ酸は、二量体化を担うアミノ酸であり得る。このような「対応する」アミノ酸または核酸は、一定範囲にわたる領域またはドメインであってもよい。従って、そのような場合、本明細書において「対応する」領域またはドメインと称される。 As used herein, a "corresponding" amino acid or nucleic acid has, or has, in a polypeptide molecule or a polynucleotide molecule, the same action as a given amino acid or nucleotide in a polypeptide or polynucleotide that serves as a reference for comparison. Amino acids or nucleotides that are expected to be predicted, especially in the case of enzyme molecules, amino acids that are present at similar positions in the active site and make similar contributions to catalytic activity. For example, if it is an antisense molecule, it can be a similar part in the ortholog corresponding to a particular part of the antisense molecule. The corresponding amino acids are specified to be, for example, cysteineized, glutathioneized, SS bond formed, oxidized (eg, methionine side chain oxidation), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. Can be an amino acid. Alternatively, the corresponding amino acid can be the amino acid responsible for dimerization. Such "corresponding" amino acids or nucleic acids may be regions or domains over a range. Thus, such cases are referred to herein as "corresponding" regions or domains.
　本明細書において「対応する」遺伝子（例えば、ポリペプチド分子またはポリヌクレオチド分子）とは、ある種において、比較の基準となる種における所定の遺伝子と同様の作用を有するか、または有することが予測される遺伝子（例えば、ポリペプチド分子またはポリヌクレオチド分子）をいい、そのような作用を有する遺伝子が複数存在する場合、進化学的に同じ起源を有するものをいう。従って、ある遺伝子に対応する遺伝子は、その遺伝子のオルソログであり得る。従って、マウス、ラットのＲＡＧＥ（または可溶性形態のｓＲＡＧＥ）は、それぞれ、ヒトにおいて、対応するＲＡＧＥ（ｓＲＡＧＥまたは可溶性形態のｓＲＡＧＥ）を見出すことができる。そのような対応する遺伝子は、当該分野において周知の技術を用いて同定することができる。したがって、例えば、ある動物（例えば、マウス）における対応する遺伝子は、対応する遺伝子の基準となる遺伝子（例えば、ＲＡＧＥまたは可溶性形態のｓＲＡＧＥ）は、ある動物の配列をクエリ配列として用いてその動物（例えばヒト、ラット）の配列データベースを検索することによって見出すことができる。 As used herein, a "corresponding" gene (eg, a polypeptide molecule or a polynucleotide molecule) has, or is expected to have, in some species, similar action to a given gene in a species of reference for comparison. A gene to be produced (for example, a polypeptide molecule or a polynucleotide molecule), and when a plurality of genes having such an action exist, those having the same evolutionary origin. Therefore, the gene corresponding to a gene can be the ortholog of that gene. Thus, mouse and rat RAGEs (or soluble forms of sRAGE) can each find a corresponding RAGE (sRAGE or soluble form of sRAGE) in humans. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, the corresponding gene in an animal (eg, mouse) is the reference gene for the corresponding gene (eg, RAGE or soluble form of sRAGE), which uses the sequence of the animal as a query sequence. For example, it can be found by searching the sequence database of humans and rats).
　本明細書において「生物学的機能」とは、ある遺伝子またはそれに関する核酸分子もしくはポリペプチドについて言及するとき、その遺伝子、核酸分子またはポリペプチドが生体内において有し得る特定の機能をいい、これには、例えば、特異的な抗体の生成、酵素活性、抵抗性の付与等を挙げることができるがそれらに限定されない。本開示においては、例えば、ＲＡＧＥがヘモペキシン等のマーカーを認識する機能などを挙げることができるがそれらに限定されない。本明細書において、生物学的機能は、「生物学的活性」によって発揮され得る。本明細書において「生物学的活性」とは、ある因子（例えば、ポリヌクレオチド、タンパク質など）が、生体内において有し得る活性のことをいい、種々の機能（例えば、転写促進活性）を発揮する活性が包含され、例えば、ある分子との相互作用によって別の分子が活性化または不活化される活性も包含される。２つの因子が相互作用する場合、その生物学的活性は、その二分子との間の結合およびそれによって生じる生物学的変化、例えば、一つの分子を抗体を用いて沈降させたときに他の分子も共沈するとき、２分子は結合していると考えられる。したがって、そのような共沈を見ることが一つの判断手法として挙げられる。例えば、ある因子が酵素である場合、その生物学的活性は、その酵素活性を包含する。別の例では、ある因子がリガンドである場合、そのリガンドが対応する受容体への結合を包含する。そのような生物学的活性は、当該分野において周知の技術によって測定することができる。 As used herein, the term "biological function" refers to a specific function that a gene, nucleic acid molecule or polypeptide can have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto. Examples include, but are not limited to, specific antibody production, enzymatic activity, and resistance imparting. In the present disclosure, for example, the function of RAGE to recognize a marker such as hemopexin can be mentioned, but the present invention is not limited thereto. As used herein, biological function can be exerted by "biological activity". As used herein, the term "biological activity" refers to the activity that a certain factor (for example, polynucleotide, protein, etc.) can have in vivo, and exerts various functions (for example, transcription promoting activity). Activities include, for example, the activity of activating or inactivating another molecule by interacting with one molecule. When two factors interact, their biological activity is the binding between the two molecules and the resulting biological changes, eg, when one molecule is precipitated with an antibody to the other. When the molecules also coprecipitate, the two molecules are considered to be bound. Therefore, seeing such coprecipitation is one of the judgment methods. For example, if a factor is an enzyme, its biological activity comprises that enzymatic activity. In another example, when a factor is a ligand, the ligand involves binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.
　したがって、「活性」は、結合（直接的または間接的のいずれか）を示すかまたは明らかにするか；応答に影響する（すなわち、いくらかの曝露または刺激に応答する測定可能な影響を有する）、種々の測定可能な指標をいい、例えば、本開示のポリペプチドまたはポリヌクレオチドに直接結合する化合物の親和性、または例えば、いくつかの刺激後または事象後の上流または下流のタンパク質の量あるいは他の類似の機能の尺度が、挙げられる。 Thus, "activity" indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus). Various measurable indicators, such as the affinity of a compound that binds directly to a polypeptide or polynucleotide of the present disclosure, or, for example, the amount of upstream or downstream protein or other after some stimulation or event. A measure of similar function can be mentioned.
　本明細書において「被験体」とは、本開示の診断または検出等の対象となる生物（例えば、ヒト）をいう。 As used herein, the term "subject" refers to an organism (for example, a human) that is the subject of the diagnosis or detection of the present disclosure.
　本明細書において「試料」とは、被験体等から得られた任意の物質をいい、例えば、体液（血液、唾液、尿、涙液、脳脊髄液等）が含まれる。 In the present specification, the "sample" refers to any substance obtained from a subject or the like, and includes, for example, body fluids (blood, saliva, urine, tears, cerebrospinal fluid, etc.).
　本明細書において「薬剤」、「剤」または「因子」（いずれも英語ではａｇｅｎｔに相当する）は、広義には、交換可能に使用され、意図する目的を達成することができる限りどのような物質または他の要素（例えば、光、放射能、熱、電気などのエネルギー）でもあってもよい。そのような物質としては、例えば、タンパク質、ポリペプチド、オリゴペプチド、ペプチド、ポリヌクレオチド、オリゴヌクレオチド、ヌクレオチド、核酸（例えば、ｃＤＮＡ、ゲノムＤＮＡのようなＤＮＡ、ｍＲＮＡのようなＲＮＡを含む）、ポリサッカリド、オリゴサッカリド、脂質、有機低分子（例えば、ホルモン、リガンド、情報伝達物質、有機低分子、コンビナトリアルケミストリで合成された分子、医薬品として利用され得る低分子（例えば、低分子リガンドなど）など）、これらの複合分子が挙げられるがそれらに限定されない。ポリヌクレオチドに対して特異的な因子としては、代表的には、そのポリヌクレオチドの配列に対して一定の配列相同性を（例えば、７０％以上の配列同一性）もって相補性を有するポリヌクレオチド、プロモーター領域に結合する転写因子のようなポリペプチドなどが挙げられるがそれらに限定されない。ポリペプチドに対して特異的な因子としては、代表的には、そのポリペプチドに対して特異的に指向された抗体またはその誘導体あるいはその類似物（例えば、単鎖抗体）、そのポリペプチドが受容体またはリガンドである場合の特異的なリガンドまたは受容体、そのポリペプチドが酵素である場合、その基質などが挙げられるがそれらに限定されない。 As used herein, "drug", "drug" or "factor" (both of which correspond to agents in English) are used interchangeably as long as they can achieve their intended purpose. It may also be a substance or other element (eg, energy such as light, radioactivity, heat, electricity). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, cDNA, DNA such as genomic DNA, RNA such as mRNA), poly. Saccharides, oligosaccharides, lipids, organic small molecules (eg, hormones, ligands, signaling substances, organic small molecules, molecules synthesized with combinatorial chemistries, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.)) , These complex molecules include, but are not limited to. Factors specific to a polynucleotide typically include polynucleotides that have certain sequence homology (eg, 70% or more sequence identity) and complementarity to the sequence of the polynucleotide. Examples include, but are not limited to, polypeptides such as transcription factors that bind to the promoter region. Factors specific for a polypeptide are typically an antibody or derivative thereof or an analog thereof (eg, a single chain antibody) specifically directed to that polypeptide, which is accepted by the polypeptide. Specific ligands or receptors in the case of a body or ligand, substrates thereof when the polypeptide is an enzyme, and the like are included, but are not limited thereto.
　本明細書において「相互作用」とは、２つの物質についていうとき、一方の物質と他方の物質との間で力（例えば、分子間力（ファンデルワールス力）、水素結合、疎水性相互作用など）を及ぼしあうこという。通常、相互作用をした２つの物質は、会合または結合している状態にある。 As used herein, the term "interaction" refers to two substances, that is, a force (for example, an intermolecular force (Van der Waals force), a hydrogen bond, a hydrophobic interaction) between one substance and the other substance. Etc.). Usually, the two interacting substances are in an associated or bound state.
　本明細書中で使用される用語「結合」は、２つのタンパク質もしくは化合物または関連するタンパク質もしくは化合物の間、あるいはそれらの組み合わせの間での、物理的相互作用または化学的相互作用を意味する。結合には、イオン結合、非イオン結合、水素結合、ファンデルワールス結合、疎水性相互作用などが含まれる。物理的相互作用（結合）は、直接的または間接的であり得、間接的なものは、別のタンパク質または化合物の効果を介するかまたは起因する。直接的な結合とは、別のタンパク質または化合物の効果を介してもまたはそれらに起因しても起こらず、他の実質的な化学中間体を伴わない、相互作用をいう。 As used herein, the term "binding" means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions and the like. Physical interactions (bindings) can be direct or indirect, the indirect being through or due to the effects of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and is not accompanied by other substantial chemical intermediates.
　本明細書中で使用される「接触（させる）」とは、化合物を、直接的または間接的のいずれかで、本開示のマーカー、リガント等として機能しうるポリペプチドまたはポリヌクレオチドに対して物理的に近接させることを意味する。ポリペプチドまたはポリヌクレオチドは、多くの緩衝液、塩、溶液などに存在させることができる。接触とは、核酸分子またはそのフラグメントをコードするポリペプチドを含む、例えば、ビーカー、マイクロタイタープレート、細胞培養フラスコまたはマイクロアレイ（例えば、遺伝子チップ）などに化合物を置くことが挙げられる。 As used herein, "contacting" means physics of a compound, either directly or indirectly, with respect to a polypeptide or polynucleotide capable of functioning as a marker, ligand, etc. of the present disclosure. It means to bring them closer to each other. The polypeptide or polynucleotide can be present in many buffers, salts, solutions and the like. Contact includes placing the compound in, for example, a beaker, a microtiter plate, a cell culture flask or a microarray (eg, a gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.
　一つの局面において、本開示は、疾患（脂質異常症、糖尿病合併症、肝疾患、および、アルツハイマー型認知症など）を診断、治療の有効性を評価するのに有用な酸化ＬＤＬまたは刺激性ＡＧＥｓを検出するために用いられる。 In one aspect, the present disclosure is an oxidized LDL or stimulating AGEs useful for diagnosing diseases (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease) and assessing the effectiveness of treatment. Is used to detect.
　本明細書において「肝疾患」とは、肝臓における任意の疾患を指す。本発明が対象とする肝疾患は、任意の肝疾患でありうるが、肝疾患は、慢性脂肪肝疾患や急性脂肪肝疾患であり得、これらは炎症性疾患であり得、あるいは生活習慣に関連した炎症性の疾患であり得るし、非アルコール性の疾患であり得るし、非ウイルス性の疾患であり得る。本発明は、非アルコール性脂肪肝（ＮＡＦＬ）および非アルコール性脂肪肝炎（ＮＡＳＨ）」の他、ＮＡＳＨがさらに進行した状態である、肝硬変および肝細胞がんなどの疾患の診断に有用でありうる。 As used herein, the term "liver disease" refers to any disease in the liver. The liver disease targeted by the present invention can be any liver disease, but the liver disease can be a chronic fatty liver disease or an acute fatty liver disease, which can be an inflammatory disease or is related to lifestyle. It can be an inflammatory disease, a non-alcoholic disease, or a non-viral disease. The present invention may be useful in diagnosing "non-alcoholic steatohepatitis (NAFL) and non-alcoholic steatohepatitis (NASH)" as well as diseases such as liver cirrhosis and hepatocellular carcinoma in which NASH is further advanced. ..
　本明細書において使用する場合、用語「慢性脂肪肝疾患」は、慢性的に肝臓に多量の脂肪が蓄積する状態をいう。慢性的とは、症状が徐々に発症して、治療および経過が長期にわたることを意味する。慢性的な疾患が生じる要因としては、年齢、性別、生活習慣、遺伝的要因、肥満、各種ホルモン異常、一部の薬剤の摂取などが挙げられるが、これらに限定されない。したがって、慢性脂肪肝疾患は、「急性」脂肪肝疾患とは全く異なる疾患であり、病態や原因なども異なり、治療法や予防法も異なるものである。 As used herein, the term "chronic fatty liver disease" refers to a condition in which a large amount of fat is chronically accumulated in the liver. Chronic means that symptoms develop gradually and treatment and course are long-term. Factors that cause chronic diseases include, but are not limited to, age, gender, lifestyle, genetic factors, obesity, various hormonal abnormalities, and intake of some drugs. Therefore, chronic fatty liver disease is a disease that is completely different from "acute" fatty liver disease, has different pathological conditions and causes, and has different treatment and prevention methods.
　本明細書において使用する場合、用語「炎症性疾患」とは、炎症を伴う疾患をいい、脂肪肝疾患について炎症性疾患というときは、炎症を伴う任意の脂肪肝疾患をいう。炎症性疾患である脂肪肝疾患には、非アルコール性脂肪肝炎（ＮＡＳＨ）およびこれに対応するアルコール性の脂肪肝炎も包含されることが理解される。 As used herein, the term "inflammatory disease" refers to a disease that accompanies inflammation, and the term "inflammatory disease" refers to any fatty liver disease that accompanies inflammation. It is understood that fatty liver disease, which is an inflammatory disease, also includes non-alcoholic steatohepatitis (NASH) and the corresponding alcoholic steatohepatitis.
　本明細書において使用する場合、用語「生活習慣に関連した炎症性の疾患」は、生活習慣を主な要因とし、炎症を伴う疾患をいう。本発明における生活習慣としては、運動量、栄養バランス、喫煙、アルコール摂取、睡眠時間等が挙げられるが、これらに限定されない。「炎症性の疾患」は、当該分野において通常使用される意味で使用され、生体の損傷に対して生じる組織の局所反応を特徴とする疾患を意味する。特定の実施形態では、本発明における炎症性の疾患は、肝臓に関連する炎症性の疾患である。 As used herein, the term "lifestyle-related inflammatory disease" refers to a disease that is accompanied by inflammation, with lifestyle as the main factor. Lifestyle-related habits in the present invention include, but are not limited to, exercise amount, nutritional balance, smoking, alcohol intake, sleep time, and the like. "Inflammatory disease" is used in the sense commonly used in the art and means a disease characterized by a local response of tissue to the injury of a living body. In certain embodiments, the inflammatory disease in the present invention is a liver-related inflammatory disease.
　本明細書において使用する場合、用語「非アルコール性の疾患」は、アルコールの摂取を主な要因としない疾患の総称である。非アルコール性の疾患に罹患した被験体には、アルコールを全く摂取しない被験体だけでなく、少量のアルコールを摂取する被験体（男性では、１日あたりに摂取される純エタノール重量が３０ｇ未満であり、女性では、１日あたりに摂取される純エタノール重量が２０ｇ未満である）も含まれる。非アルコール性疾患は、非アルコール性脂肪肝疾患（ＮＡＦＬＤ）とも総称され、非アルコール性疾患の代表的なものとしては、非アルコール性脂肪肝（ＮＡＦＬ）や非アルコール性脂肪肝炎（ＮＡＳＨ）が挙げられる。 As used herein, the term "non-alcoholic disease" is a general term for diseases whose main factor is alcohol intake. Subjects with non-alcoholic diseases include not only subjects who do not consume alcohol at all, but also subjects who consume a small amount of alcohol (in men, the weight of pure ethanol consumed per day is less than 30 g). Yes, in women, the weight of pure ethanol ingested per day is less than 20 g). Non-alcoholic diseases are also collectively referred to as non-alcoholic steatohepatitis (NAFLD), and typical non-alcoholic diseases include non-alcoholic steatohepatitis (NAFL) and non-alcoholic steatohepatitis (NASH). Be done.
　本明細書において使用する場合、用語「非アルコール性脂肪肝（ＮＡＦＬ）」は、当該分野において通常使用される意味で使用され、非アルコール性であり、肝臓に脂肪が蓄積することを特徴とするが、肝臓への炎症細胞の浸潤を認めない疾患を意味する。ＮＡＦＬは、脂肪肝疾患の中でも、比較的症状が軽く予後が良好な疾患であるが、ＮＡＳＨまたは肝硬変などの、より重症の疾患へと進展する可能性がある。非アルコール性脂肪肝とは対照的に、アルコールの摂取を主な要因とする、「アルコール性脂肪肝」という疾患も存在する。非アルコール性脂肪肝とアルコール性脂肪肝とは、アルコールの摂取の有無の点で異なるが、これらの脂肪肝の病態は類似しており、罹患後２０年経過すると、これらの差異はなくなることも報告されている。 As used herein, the term "non-alcoholic fatty liver (NAFL)" is used in the sense commonly used in the art and is characterized by non-alcoholic and accumulation of fat in the liver. However, it means a disease in which inflammatory cells do not infiltrate the liver. NAFL is a disease with relatively mild symptoms and a good prognosis among fatty liver diseases, but it may progress to more severe diseases such as NASH or cirrhosis. In contrast to non-alcoholic fatty liver, there is also a disease called "alcoholic fatty liver" whose main factor is alcohol intake. Non-alcoholic fatty liver and alcoholic fatty liver differ in the presence or absence of alcohol intake, but the pathophysiology of these fatty livers is similar, and these differences may disappear 20 years after illness. It has been reported.
　本明細書において使用する場合、用語「非アルコール性脂肪肝炎（ＮＡＳＨ）」は、当該分野において通常使用される意味で使用され、肝細胞における脂肪蓄積、肝細胞風船様変性（バルーニング変性）、アポトーシスの増加、肝臓の中心静脈領域への炎症性細胞の浸潤、脂肪変性した肝細胞をマクロファージが取り囲み貪食・処理する現象（ｈＣＬＳ）、肝臓における過剰な細胞外基質の沈着を伴う肝臓の疾患を意味する。 As used herein, the term "non-alcoholic steatosis (NASH)" is used in the sense commonly used in the art, including fat accumulation in hepatocytes, hepatocyte balloon-like degeneration (baluning degeneration), and apoptosis. Infiltration of inflammatory cells into the central venous region of the liver, a phenomenon in which macrophages surround and phagocytose and process fatty degenerated hepatocytes (hCLS), and liver diseases accompanied by excessive deposition of extracellular matrix in the liver. To do.
　本明細書において使用する場合、用語「Ｍａｔｔｅｏｎｉ分類」は、当該分野において通常使用される意味で使用され、脂肪肝疾患の状態に基づいて脂肪肝疾患の重症度を判断する分類法である。Ｍａｔｔｅｏｎｉ分類は、肝組織における（１）肝細胞の脂肪変性（ｓｔｅａｔｏｓｉｓ）、（２）炎症性細胞浸潤（ｉｎｆｌａｍｍａｔｉｏｎ）、（３）肝細胞の風船様腫大（ｂａｌｌｏｏｎｉｎｇｈｅｐａｔｏｃｙｔｅ）、（４）肝臓の線維化（ｆｉｂｒｏｓｉｓ）、および（５）Ｍａｌｌｏｒｙ－Ｄｅｎｋ体の有無に基づいて、ＮＡＦＬＤをＴｙｐｅ１～Ｔｙｐｅ４の４種類に分類する手法である。Ｍａｔｔｅｏｎｉ　Ｔｙｐｅ　１は肝細胞の脂肪変性のみ、Ｔｙｐｅ　２は肝細胞の脂肪変性に炎症細胞の浸潤のみを認めるもの、Ｔｙｐｅ　３は肝細胞の風船様腫大を認めるもの、Ｔｙｐｅ　４はＴｙｐｅ　３に加えて肝線維化を認めるもの、と定義されており、Ｔｙｐｅ　３および４がＮＡＳＨと診断される。 As used herein, the term "Mattenoni classification" is used in the sense commonly used in the art and is a classification method for determining the severity of fatty liver disease based on the condition of fatty liver disease. The Mattenoni classification includes (1) hepatocyte steatosis, (2) inflammatory cell infiltration, (3) hepatocyte balloon-like swelling, and (4) liver fibers. It is a method of classifying NAFLD into four types, Type 1 to Type 4, based on the presence or absence of fibrosis and (5) Mallory-Denk form. Matteni Type 1 shows only hepatocyte fat degeneration, Type 2 shows only hepatocyte fat degeneration with infiltration of inflammatory cells, Type 3 shows hepatocyte balloon-like swelling, and Type 4 is added to Type 3. Hepatocytes 3 and 4 are diagnosed as NASH.
　本明細書において使用する場合、用語「肝臓の線維化」とは、肝臓の修復または反応プロセスにおいて、過剰な線維性結合組織が形成することをいう。線維化自体は症状を引き起こさないが、重度に線維化が進展すると、肝硬変および合併症などを引き起こし、結果として症状が発症する。線維化が多数の種類があり、原因も多数存在する。例えば、ＮＡＳＨでは、タイプ４にまで進展すると線維化がみられる。この段階になると、肝硬変や肝がんへの進行のリスクにも配慮しなければならない。また、化学物質（例えば、四塩化炭素など）によっても線維化が生じる（急性の線維化と表現することができる。）が、肝臓への脂肪の蓄積が認められず、本発明の慢性脂肪肝疾患における線維化（慢性の線維化と表現することができる）とは性質を異にする。理論に束縛されることを望まないが、急性の線維化と慢性の線維化とは、病理学的態様がまったく異なるため、その予防や治療についても全く異なる病態であるといえ、得られた知見を予防薬や治療薬の開発において相互に参照することはできない。 As used herein, the term "liver fibrosis" refers to the formation of excess fibrous connective tissue during the repair or reaction process of the liver. Fibrosis itself does not cause symptoms, but when fibrosis progresses severely, it causes liver cirrhosis and complications, and as a result, symptoms develop. There are many types of fibrosis and many causes. For example, in NASH, fibrosis is seen when it progresses to type 4. At this stage, the risk of developing cirrhosis and liver cancer must also be considered. Fibrosis is also caused by chemical substances (for example, carbon tetrachloride) (which can be expressed as acute fibrosis), but no fat accumulation in the liver is observed, and the chronic fatty liver of the present invention is not observed. It is different in nature from fibrosis in disease (which can be described as chronic fibrosis). Although I do not want to be bound by theory, it can be said that acute fibrosis and chronic fibrosis are completely different pathological conditions in terms of their prevention and treatment because their pathological aspects are completely different. Cannot be referred to each other in the development of prophylactic and therapeutic agents.
　肝細胞と置き換わった線維化組織は、肝細胞の機能を有さない。さらに、線維化組織は、肝臓への血流と肝臓内での血流を妨げて、肝細胞への血液の供給を制限し、肝細胞の死滅をもたらし、それによってさらなる線維化の進展をもたらし得る。本発明は、このような線維化も予測診断することができる。 The fibrotic tissue that replaced the hepatocytes does not have the function of hepatocytes. In addition, fibrotic tissue impedes blood flow to and in the liver, limiting the supply of blood to hepatocytes and leading to hepatocyte death, thereby leading to further development of fibrosis. obtain. The present invention can also predictively diagnose such fibrosis.
　本明細書において「診断」とは、被験体における疾患、障害、状態などに関連する種々のパラメータを同定し、そのような疾患、障害、状態の現状または未来を判定することをいう。本開示の方法、装置、システムを用いることによって、体内の状態を調べることができ、そのような情報を用いて、被験体における疾患、障害、状態、投与すべき処置または予防のための処方物または方法などの種々のパラメータを選定することができる。本明細書において、狭義には、「診断」は、現状を診断することをいうが、広義には「予測診断」「事前診断」等を含む。早期の診断は「早期診断」ということもある。 As used herein, the term "diagnosis" refers to identifying various parameters related to a disease, disorder, condition, etc. in a subject and determining the current state or future of such a disease, disorder, condition. By using the methods, devices, and systems of the present disclosure, the condition within the body can be investigated and such information can be used to formulate a disease, disorder, condition, treatment to be administered or prevention in the subject. Alternatively, various parameters such as a method can be selected. In the present specification, in a narrow sense, "diagnosis" means diagnosing the current state, but in a broad sense, it includes "predictive diagnosis", "pre-diagnosis" and the like. Early diagnosis is sometimes called "early diagnosis".
　本明細書において特に、「予測診断」または「事前診断」とは、交換可能に使用され、ＬＤＬまたはＡＧＥｓを認識することができる分子（ＣＴＬＤ１４、ｓＲＡＧＥ等）を用いて、肝疾患、糖尿病、糖尿病性腎症、糖尿病性網膜症、糖尿病性神経症等の糖尿病合併症等について言及する場合、糖尿病、糖尿病性腎症、糖尿病性網膜症、糖尿病性神経症等の糖尿病合併症等の発症前の段階を検出することをいい、将来の発症リスクを判定すること、肝疾患、糖尿病、糖尿病性腎症、糖尿病性網膜症、糖尿病性神経症等の糖尿病合併症等の予防を目的として糖尿病に罹患するおそれの有無を判定することを含む。本開示の方法、キット、組成物、検出剤、診断剤、システム等を用いることによって、体内の状態を事前に調べることができ、そのような情報を用いて、被験体における疾患、障害、状態、投与すべき処置または予防のための処方物または方法などの種々のパラメータを選定することができる。本明細書では、「予測診断」または「事前診断」は、他の従来の方法では診断できない段階での診断をも包含することから、「早期診断」の概念と一部重なって用いられる。 In particular, as used herein, "predictive diagnosis" or "pre-diagnosis" refers to liver disease, diabetes, diabetes using molecules that are interchangeably used and capable of recognizing LDL or AGEs (CTLD14, sRAGE, etc.). When referring to diabetic complications such as diabetic nephropathy, diabetic nephropathy, diabetic nephropathy, etc., before the onset of diabetic complications such as diabetes, diabetic nephropathy, diabetic nephropathy, diabetic nephropathy, etc. Detecting the stage, determining the risk of developing the disease in the future, and suffering from diabetes for the purpose of preventing diabetic complications such as liver disease, diabetes, diabetic nephropathy, diabetic nephropathy, and diabetic neuropathy. Includes determining if there is a risk of By using the methods, kits, compositions, detection agents, diagnostic agents, systems, etc. of the present disclosure, the condition in the body can be investigated in advance, and such information can be used to determine the disease, disorder, condition in the subject. Various parameters can be selected, such as the procedure or method for treatment or prevention to be administered. In the present specification, "predictive diagnosis" or "pre-diagnosis" is used in part with the concept of "early diagnosis" because it also includes diagnosis at a stage that cannot be diagnosed by other conventional methods.
　本開示の診断方法は、原則として、身体から出たものを利用することができることから、医師などの医療従事者の手を離れて実施することができることから、産業上有用である。本明細書において、医師などの医療従事者の手を離れて実施することができることを明確にするために、特に「予測診断、事前診断もしくは診断」を「支援」すると称することがある。広義には、「診断」には、治療の有効性を評価することも包含される。 As a general rule, the diagnostic method of the present disclosure is industrially useful because it can be used from the body and can be carried out without the hands of medical professionals such as doctors. In the present specification, in particular, "predictive diagnosis, pre-diagnosis or diagnosis" may be referred to as "support" in order to clarify that it can be carried out without the hands of medical personnel such as doctors. In a broad sense, "diagnosis" also includes assessing the effectiveness of treatment.
　本明細書において使用される場合、用語「検出剤」とは、広義には、目的の物質（例えば、疾患に関連する酸化ＬＤＬまたは刺激性ＡＧＥｓなど）を検出できるあらゆる因子をいう。 As used herein, the term "detector" broadly refers to any factor capable of detecting a substance of interest (eg, oxidized LDL or irritant AGEs associated with a disease).
　本明細書において使用される場合、用語「診断剤」とは、広義には、目的の状態（例えば、疾患（脂質異常症、糖尿病合併症、肝疾患、および、アルツハイマー型認知症など）など）を診断できるあらゆる因子をいう。 As used herein, the term "diagnostic agent" is broadly defined as a condition of interest (eg, disease (eg, dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease, etc.)). Any factor that can diagnose.
　本明細書において、「測定」とは、当該分野において使用される通常の意味で用いられ、ある対象について、量がどれほどか測って求めることをいう。本明細書において「検出」とは、当該分野において使用される通常の意味で用いられ、物質や成分等を検査して見つけだすことをいい、「同定」とは、ある対象について、そのものにかかわる既存の分類のなかからそれの帰属先をさがす行為をいい、化学分野において使用される場合、対象となる物質の化学物質としての同一性を決定する（例えば、化学構造を決定する）ことをいい、「定量」とは、対象となる物質の存在する量を決定することをいう。本明細書において「（分子）とのコンジュゲート形成により検出または定量」とは、検出または定量の目的となる対象の検出または定量が、その対象と別の実体（ｅｎｔｉｔｙ）がコンジュゲートを形成するかどうかを指標になされることをいう。コンジュゲート形成を指標にする場合と、コンジュゲート形成の阻害（競合分子を用いる）を指標にする場合とがあり得る。 In the present specification, "measurement" is used in the usual sense used in the art, and means to measure and obtain the amount of a certain object. In the present specification, "detection" is used in the usual meaning used in the art, and means to inspect and find a substance, a component, etc., and "identification" is an existing substance related to a certain object. When used in the field of chemistry, it means determining the identity of the target substance as a chemical substance (for example, determining the chemical structure). "Quantitative" means determining the amount of a substance of interest present. In the present specification, "detection or quantification by formation of a conjugate with (molecule)" means that the detection or quantification of an object to be detected or quantified forms a conjugate with an entity different from the object. It means that whether or not it is used as an index. There may be a case where conjugate formation is used as an index and a case where inhibition of conjugate formation (using a competitive molecule) is used as an index.
　本明細書において「治療」とは、ある疾患または障害について、そのような状態になった場合に、そのような疾患または障害の悪化を防止、好ましくは、現状維持、より好ましくは、軽減、さらに好ましくは消退させることをいう。 As used herein, the term "treatment" refers to a disease or disorder that, when such a condition occurs, prevents the exacerbation of the disease or disorder, preferably maintains the status quo, more preferably alleviates, and further. Preferably, it means to withdraw.
　本明細書において「予防」とは、ある疾患（脂質異常症、糖尿病合併症、肝疾患、および、アルツハイマー型認知症など）または障害について、そのような状態になる前に、そのような状態にならないようにすることをいう。本開示の予測診断または事前診断を行うことによって、酸化ＬＤＬまたは刺激性ＡＧＥｓに関連する疾患または障害等の予防をするか、あるいは予防のための対策を講じることができる。本開示の予測診断または事前診断を行うことによって、糖尿病性腎症、糖尿病性網膜症、糖尿病性神経症等の糖尿病合併症等の予防をするか、あるいは予防のための対策を講じることもできる。 As used herein, the term "prevention" refers to a disease (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease) or disorder before it becomes such a condition. It means not to become. By performing the predictive diagnosis or pre-diagnosis of the present disclosure, it is possible to prevent diseases or disorders related to oxidized LDL or irritating AGEs, or to take preventive measures. By performing the predictive diagnosis or pre-diagnosis of the present disclosure, it is possible to prevent diabetic complications such as diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy, or to take measures for prevention. ..
　（好ましい実施形態）
　以下に好ましい実施形態の説明を記載するが、この実施形態は本開示の例示であり、本開示の範囲はそのような好ましい実施形態に限定されないことが理解されるべきである。当業者はまた、以下のような好ましい実施例を参考にして、本開示の範囲内にある改変、変更などを容易に行うことができることが理解されるべきである。これらの実施形態について、当業者は適宜、任意の実施形態を組み合わせ得る。 (Preferable embodiment)
Although a description of preferred embodiments will be described below, it should be understood that this embodiment is an example of the present disclosure and the scope of the present disclosure is not limited to such preferred embodiments. It should be understood that those skilled in the art can also easily make modifications, changes, etc. within the scope of the present disclosure with reference to the following preferred embodiments. For these embodiments, those skilled in the art may optionally combine any embodiments.
　（デバイスまたはキット）
　一局面において、本開示は、毛管現象による試料の展開を行うメンブレンを含む、生体マーカー分子の異常型を該生体マーカー分子とのコンジュゲート形成により検出または定量するためのデバイスまたはキットであって、該メンブレンが、生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部とを含み、該キットまたはデバイスは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含む、キットまたはデバイスを提供する。 (Device or kit)
In one aspect, the present disclosure is a device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule, including a membrane that develops a sample by capillary phenomenon. The membrane specifically binds to a detection unit containing a detection binder that specifically binds to a biomarker molecule or a competing molecule thereof, and a binding molecule having an abnormal form of the biomarker molecule or a binding molecule capable of forming a conjugate with the competing molecule thereof. The kit or device includes a control part containing a control binder to be bound, and the kit or device includes a sample contact part and fluorescent nanoparticles as a part or a separate element of a membrane.
　いくつかの実施形態において、メンブレンが、上流から下流にかけて、検出部およびコントロール部をこの順番で含み得る。 In some embodiments, the membrane may include a detection unit and a control unit in this order from upstream to downstream.
　いくつかの実施形態において、試料接触部、検出部、およびコントロール部が、相互に毛管現象で試料が浸透するように配置または連結され得る。 In some embodiments, the sample contact, detection, and control units may be arranged or connected to each other so that the sample penetrates into each other by capillarity.
　いくつかの実施形態において、生体マーカー分子はＬＤＬまたはＡＧＥｓであり得、生体マーカー分子の異常型は変性ＬＤＬまたは刺激性ＡＧＥｓであり得る。 In some embodiments, the biomarker molecules can be LDL or AGEs, and the aberrant forms of the biomarker molecules can be denatured LDL or stimulating AGEs.
　いくつかの実施形態において、結合分子はＣＴＬＤ１４またはｓＲＡＧＥであり得る。 In some embodiments, the binding molecule can be CTLD14 or sRAGE.
　いくつかの実施形態において、検出対象が、変性ＬＤＬである場合は、検出用結合剤は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメントであり得、検出対象が刺激性ＡＧＥｓである場合は、抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントであり得る。 In some embodiments, if the detection target is a denatured LDL, the detection binding agent can be an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, and the detection target is irritating. When it is AGEs, it can be an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof.
　いくつかの実施形態において、本開示のキットまたはデバイスは、生体マーカー分子の競合分子をさらに含んでもよいし、含まなくてもよい。 In some embodiments, the kits or devices of the present disclosure may or may not further include competing molecules of biomarker molecules.
　特定の実施形態において、生体マーカー分子はＬＤＬであり、生体マーカー分子の異常型は変性ＬＤＬであり、結合分子はＣＴＬＤ１４であり、検出用結合剤は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメントであり得る。 In certain embodiments, the biomarker molecule is LDL, the variant of the biomarker molecule is denatured LDL, the binding molecule is CTLD14, and the detection binding agent is an anti-LDL antibody, anti-denatured LDL antibody or anti-ApoB antibody. Or they can be antigen-binding fragments.
　特定の実施形態において、前記生体マーカー分子はＡＧＥｓであり、前記生体マーカー分子の異常型は刺激性ＡＧＥｓであり、前記結合分子はｓＲＡＧＥであり、前記検出用結合剤は抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントであり、前記キットまたはデバイスは、前記生体マーカー分子の競合分子をさらに含み、該競合分子はＧ－ＢＳＡもしくはＧ－ＯＶＡであり得る。 In certain embodiments, the biomarker molecule is AGEs, the variant of the biomarker molecule is stimulating AGEs, the binding molecule is sRAGE, and the detection binding agent is an anti-BSA antibody or anti-OVA antibody. Or an antigen-binding fragment thereof, the kit or device further comprises a competing molecule of the biomarker molecule, which competing molecule can be G-BSA or G-OVA.
　いくつかの実施形態において、蛍光ナノ粒子は、検出試薬として提供され得る。検出試薬は、本開示のデバイスまたはキットとは別個に提供されてもよいし、本開示のデバイスまたはキットと一体となって提供されてもよい。検出試薬は、試料と混合して使用してもよいし、コンジュゲート部に含めてもよい。検出試薬が試料と混合される場合、コンジュゲート部は省略されてもよい。試料を水平方向に展開する場合は、メンブレンは検出試薬を含むコンジュゲート部を備えているのが好ましく、試料を垂直方向に展開する場合は、コンジュゲート部を省略し、検出試薬を試料中にあらかじめ混合しておくのが好ましい。 In some embodiments, the fluorescent nanoparticles can be provided as a detection reagent. The detection reagents may be provided separately from the devices or kits of the present disclosure, or may be provided together with the devices or kits of the present disclosure. The detection reagent may be mixed with the sample and used, or may be included in the conjugate portion. When the detection reagent is mixed with the sample, the conjugate section may be omitted. When the sample is developed in the horizontal direction, the membrane preferably has a conjugate part containing the detection reagent, and when the sample is developed in the vertical direction, the conjugate part is omitted and the detection reagent is contained in the sample. It is preferable to mix them in advance.
　（組成物）
　別の局面において、本開示は、蛍光ナノ粒子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部とを含み、該蛍光ナノ粒子は、該結合分子を標識することにより使用される、組成物を提供する。いくつかの実施形態において、デバイス、システムまたはキットは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含み得る。 (Composition)
In another aspect, the present disclosure is a composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising fluorescent nanoparticles, said device, system or kit. Includes a membrane that develops a sample by capillary phenomenon, and the membrane contains a detection binder that specifically binds to a biomarker molecule or a competing molecule thereof, and an abnormal form of the biomarker molecule or its competition. Provided is a composition comprising a control moiety containing a control binder that specifically binds to a molecule and a binding molecule capable of forming a conjugate, wherein the fluorescent nanoparticles are used by labeling the binding molecule. To do. In some embodiments, the device, system or kit may include the sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
　さらなる局面において、本開示は、蛍光ナノ粒子で標識された結合分子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、該結合分子は、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有し、該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と、該結合分子に特異的に結合するコントロール用結合剤を含むコントロール部とを含む、組成物を提供する。いくつかの実施形態において、デバイス、システムまたはキットは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含み得る。 In a further aspect, the disclosure is a composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising a binding molecule labeled with fluorescent nanoparticles. The binding molecule has the ability to form a conjugate with an atypical form of the biomarker molecule or a competing molecule thereof, the device, system or kit comprises a membrane that develops the sample by capillary phenomenon, and the membrane is the biomarker molecule. Alternatively, a composition comprising a detection unit containing a detection binder that specifically binds to the competing molecule and a control unit containing a control binder that specifically binds to the binding molecule is provided. In some embodiments, the device, system or kit may include the sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
　（変性ＬＤＬ検出システム）
　一局面において、本開示は、毛管現象による試料の展開を行うメンブレンを含む、変性ＬＤＬを検出または定量するためのシステムまたはデバイスであって、該メンブレンが、蛍光ナノ粒子で標識されたＣＴＬＤ１４を含むコンジュゲート部と、抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメントを含む検出部と、ＣＴＬＤ１４に対する結合分子を含むコントロール部とを含む、システムを提供する。いくつかの実施形態において、システムまたはデバイスは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含み得る。本開示のシステムは、従来の方法と比べて、簡易かつ迅速に変性ＬＤＬを検出することが可能である。また、夾雑物を多く含む血液試料（例えば、全血、血清、血漿）などの生体試料を用いて検出することが可能である。本開示のシステムは、ラテラルフローアッセイ形式のシステムであり得る。本開示のシステムは、夾雑物を多く含む血液試料において、夾雑物を取り除くための追加の工程を必要とすることなく変性ＬＤＬを検出することが可能であるため、有利である。 (Denatured LDL detection system)
In one aspect, the present disclosure is a system or device for detecting or quantifying modified LDL that comprises a membrane that develops a sample by capillary phenomenon, wherein the membrane comprises CTLD14 labeled with fluorescent nanoparticles. Provided is a system including a conjugate part, a detection part containing an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, and a control part containing a binding molecule to CTLD14. In some embodiments, the system or device may include sample contacts and fluorescent nanoparticles as part of or as a separate element of the membrane. The system of the present disclosure can detect denatured LDL easily and quickly as compared with the conventional method. Further, it can be detected by using a biological sample such as a blood sample containing a large amount of impurities (for example, whole blood, serum, plasma). The system of the present disclosure can be a lateral flow assay type system. The system of the present disclosure is advantageous because it is possible to detect denatured LDL in a contaminating blood sample without the need for additional steps to remove the contaminants.
　いくつかの実施形態において、コンジュゲート部は、メンブレンとは別要素として提供されていてもよい。 In some embodiments, the conjugate portion may be provided as a separate element from the membrane.
　本開示の試料接触部は、試料（例えば、血液等）が接触され得る部分であり、試料が接触する形状であればどのような形状であってもよく、材質もまた、どのような材質であってもよいが、試料と反応をする材質は可能であれば回避され得る。試料接触部は、メンブレンの一部として提供されてもよく、別要素として提供されていてもよいが、いずれにしてもコントロール部および検出部と毛管現象で試料が浸透するように連結されている必要がある。 The sample contact portion of the present disclosure is a portion to which a sample (for example, blood, etc.) can be contacted, and may have any shape as long as the sample comes into contact with the sample, and the material may be any material. There may be, but materials that react with the sample can be avoided if possible. The sample contact portion may be provided as a part of the membrane or as a separate element, but in any case, the sample contact portion is connected to the control portion and the detection portion so as to penetrate the sample by capillarity. There is a need.
　いくつかの実施形態において、試料接触部は、血球分離フィルターを含み得る。血球分離フィルターとは、赤血球、白血球および血小板をろ過して、血球以外の成分をメンブレンに送り出すためのフィルターを指し、例えば、ＦＵＳＩＯＮ５、ＬＦ１、ＭＦ１、およびＶＦ２などが挙げられるが、これらに限定されない。好ましい実施形態において、血球分離フィルターは、ＦＵＳＩＯＮ５またはＬＦ１であり得る。 In some embodiments, the sample contact may include a blood cell separation filter. The blood cell separation filter refers to a filter for filtering red blood cells, white blood cells, and platelets and sending out components other than blood cells to the membrane, and examples thereof include, but are not limited to, FUSION5, LF1, MF1, and VF2. .. In a preferred embodiment, the blood cell separation filter can be FUSION 5 or LF1.
　本開示におけるコンジュゲート部は、蛍光ナノ粒子標識ＣＴＬＤ１４を含むものである。コンジュゲート部は、蛍光ナノ粒子標識ＣＴＬＤ１４と、試料とが接触可能な構造および材質で構成され得る。 The conjugate portion in the present disclosure includes a fluorescent nanoparticle-labeled CTLD14. The conjugate portion may be composed of a structure and a material in which the fluorescent nanoparticle labeled CTLD14 and the sample can be contacted.
　いくつかの実施形態において、蛍光ナノ粒子は、化合物半導体ナノ粒子や蛍光ラテックス粒子から選択され得、好ましくは、非晶質のシリカ粒子であり、最も好ましくは、粒子表面が親水性の高いシラノール基で覆われた親水性が高く、疎水性相互作用による吸着が起こり難い蛍光シリカナノ粒子（例えばＱｕａｒｔｚ　Ｄｏｔ）である。蛍光ナノ粒子標識ＣＴＬＤ１４は、コンジュゲート部に配置される。いくつかの実施形態において、ＣＴＬＤ１４は、ビオチン化、Ｈｉｓタグ付加、Ｍｙｃタグ付加、Ｆｌａｇタグ付加、Ｅタグ付加、またはＳｔｒｅｐタグ付加されており、それぞれの場合において、ＣＴＬＤ１４に対する結合分子は、ストレプトアビジン、抗Ｈｉｓ抗体、抗Ｍｙｃ抗体、抗Ｆｌａｇ抗体、抗Ｅタグ抗体、またはＳｔｒｅｐ－Ｔａｃｔｉｎであり得る。 In some embodiments, the fluorescent nanoparticles can be selected from compound semiconductor nanoparticles or fluorescent latex particles, preferably amorphous silica particles, most preferably silanol groups with a highly hydrophilic particle surface. Fluorescent silica nanoparticles (for example, Quartz Dot) that are covered with and have high hydrophilicity and are unlikely to be adsorbed by hydrophobic interaction. The fluorescent nanoparticle labeled CTLD14 is arranged in the conjugate section. In some embodiments, CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strept-tagged, in which case the binding molecule to CTLD14 is streptavidin. , Anti-His antibody, anti-Myc antibody, anti-Flag antibody, anti-E-tag antibody, or Strept-Tactin.
　いくつかの実施形態において、ＣＴＬＤ１４は、カイコ型糖鎖を有し得る。このようなカイコ型糖鎖を有するＣＴＬＤ１４は、カイコにより産生されるものであってもよく、人工的に糖鎖を付加されたものであってもよいが、カイコにより産生されるのが好ましい。ビオチンリガーゼを共発現可能に組み込んだカイコにより産生されるＣＴＬＤ１４は、ＣＴＬＤ１４の配列内にビオチン化タグを含めることによって、ビオチン化され得る。ビオチン化タグとしては、ビオチン化を受けるタグ配列としては、ＢｉｏＥａｓｅ．ｔａｇ、Ａｖｉ．ｔａｇ、ビオチン化を受け得る任意の配列が挙げられるが、これらに限定されない。ビオチン化ＣＴＬＤ１４を使用することによって、コントロール部に比較的容易に利用可能なストレプトアビジンを使用することが可能である。得られるビオチン化ＣＴＬＤ１４は、アルカリ条件下での蛍光ナノ粒子とのコンジュゲートでも安定であり、優れている。特定の実施形態において、ＣＴＬＤ１４は、配列番号２のアミノ酸配列と少なくとも約９０％、少なくとも約９５％、少なくとも約９７％、少なくとも約９８％、少なくとも約９９％、約１００％の同一性を有し得る。カイコ型糖鎖を有するビオチン化ＣＴＬＤ１４の産生方法は、参照によって本明細書に組み込まれる国際公開第２０１６／０５１８０８号にも詳述されている。 In some embodiments, CTLD14 may have silk moth-type sugar chains. CTLD14 having such a silk moth type sugar chain may be produced by silk moth or may be artificially added with a sugar chain, but it is preferably produced by silk moth. CTLD14 produced by silk moths co-expressing biotin ligase can be biotinylated by including a biotinylated tag within the sequence of CTLD14. As the biotinylated tag, the tag sequence to be biotinylated is BioEase. tag, Avi. Examples include, but are not limited to, tag, any sequence capable of undergoing biotinylation. By using biotinylated CTLD14, it is possible to use streptavidin, which is relatively easily available, in the control section. The obtained biotinylated CTLD14 is stable and excellent even when conjugated with fluorescent nanoparticles under alkaline conditions. In certain embodiments, CTLD14 has at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, about 100% identity with the amino acid sequence of SEQ ID NO: 2. obtain. A method for producing biotinylated CTLD14 having a silk moth-type sugar chain is also described in detail in WO 2016/051808, which is incorporated herein by reference.
　本開示のシステムまたはデバイスにおいて、検出部（テストスポットまたはライン）は、抗変性ＬＤＬ抗体、抗ＬＤＬ抗体また抗ＡｐｏＢ抗体を含む。検出部は、これらの抗体を介して、目的とする成分（例えば、変性ＬＤＬ、刺激性ＡＧＥｓ）が試料中に存在するかどうかの検出を可能にする部分である。したがって、検出を可能にする形状および材質であれば、どのようなもので構成されていてもよい。 In the systems or devices of the present disclosure, the detector (test spot or line) comprises an anti-denatured LDL antibody, an anti-LDL antibody or an anti-ApoB antibody. The detection unit is a part that enables detection of whether or not a target component (for example, denatured LDL, stimulating AGEs) is present in a sample via these antibodies. Therefore, any shape and material that enables detection may be used.
　本開示のシステムまたはデバイスにおいて、コントロール部（コントロールスポットまたはライン）は、デバイス上で試料が展開されたことを確認する部分であり、ＣＴＬＤ１４に対する結合分子（例えば、ＣＴＬＤ１４に対する抗体、またはＣＴＬＤ１４がビオチン化されている場合、このビオチンを検出するストレプトアビジン）を含む。検出部（テストスポットまたはライン）は、ＣＴＬＤ１４に対する結合分子がＣＴＬＤ１４に結合し、次いであるいは並行して抗体と相互作用することで、蛍光ナノ粒子標識ＣＴＬＤ１４が凝集して蛍光強度が増し、目的成分の検出または定量を可能にする。したがって、結合、制御および検出を可能にする形状および材質であれば、どのようなもので構成されていてもよい。検出または定量については、（検出または定量方法）においても詳述されている。 In the systems or devices of the present disclosure, the control section (control spot or line) is the part that confirms that the sample has been developed on the device and that the binding molecule to CTLD14 (eg, antibody to CTLD14, or biotinylated CTLD14). If so, it contains streptavidin) that detects this biotin. In the detection unit (test spot or line), the molecule binding to CTLD14 binds to CTLD14 and then or in parallel with the antibody, so that the fluorescent nanoparticle-labeled CTLD14 aggregates to increase the fluorescence intensity and the target component. Allows detection or quantification. Therefore, it may be made of any shape and material that enables binding, control and detection. Detection or quantification is also described in detail in (Detection or Quantification Method).
　（刺激性ＡＧＥｓ検出システム）
　別の局面において、本開示は、毛管現象による試料の展開を行うメンブレンを含む、刺激性ＡＧＥｓを検出または定量するためのシステムであって、該メンブレンが、蛍光ナノ粒子標識ｓＲＡＧＥを含むコンジュゲート部と、抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントを含む検出部と、ｓＲＡＧＥに対する結合分子を含むコントロール部とを含む、システムを提供する。いくつかの実施形態において、コンジュゲート部はさらにＧ－ＢＳＡもしくはＧ－ＯＶＡを含み得る。いくつかの実施形態において、コンジュゲート部は、メンブレンとは別要素として提供されていてもよい。 (Irritation AGEs detection system)
In another aspect, the present disclosure is a system for detecting or quantifying stimulating AGEs, which comprises a membrane that develops a sample by capillarity, wherein the membrane comprises a fluorescent nanoparticle labeled sRAGE. A system comprising a detection unit containing an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and a control unit containing a binding molecule to sRAGE. In some embodiments, the conjugate portion may further comprise a G-BSA or G-OVA. In some embodiments, the conjugate portion may be provided as a separate element from the membrane.
　理論に束縛されることを望まないが、例えば、ｓＲＡＧＥに対する競合により、テストライン上のスポットの濃さ（蛍光強度）が減少することにより、試料中の刺激性ＡＧＥｓを検出することができる。例えばテストラインに、抗ＢＳＡ抗体をスポットした系の場合：グルコース修飾ＢＳＡ（Ｇ－ＢＳＡ活性の弱いＡＧＥｓ）を含む反応バッファーに、蛍光ナノ粒子標識ｓＲＡＧＥと試料を添加し、試料中に刺激性ＡＧＥｓが存在しない場合、蛍光ナノ粒子標識ｓＲＡＧＥがＧ－ＢＳＡと結合し、テストラインで抗体に捕捉され凝集するため蛍光を発するのに対し、試料中に刺激性ＡＧＥｓが存在する場合、Ｇ－ＢＳＡのｓＲＡＧＥへの結合量が低下し、その結果、テストラインでの蛍光ナノ粒子標識ｓＲＡＧＥの凝集が生じ難くなり、結果的にテストスポットの蛍光が低下することが企図される。このテストラインの蛍光強度の阻害の程度で刺激性ＡＧＥｓの存在を検出することができる。テストラインに抗Ｇ－ＢＳＡ抗体を使用する場合、コンジュゲートバッファーやブロッキングバッファーにＢＳＡが使用できないことから、適宜、カゼイン、ＰＶＡなどを使用することができる。別の例として、テストラインに抗ＯＶＡ（オボアルブミン）抗体を使用し、競合用ＡＧＥｓとして、Ｇ－ＯＶＡを使用する系も実施することができる。 Although not bound by theory, for example, stimulating AGEs in a sample can be detected by reducing the spot density (fluorescence intensity) on the test line due to competition for sRAGE. For example, in the case of a system in which an anti-BSA antibody is spotted on a test line: Fluorescent nanoparticle-labeled sRAGE and a sample are added to a reaction buffer containing glucose-modified BSA (AGEs with weak G-BSA activity), and stimulating AGEs are added to the sample. In the absence of, the fluorescent nanoparticle-labeled sRAGE binds to G-BSA and fluoresces because it is captured and aggregated by the antibody on the test line, whereas in the presence of stimulating AGEs in the sample, G-BSA It is intended that the amount of binding to sRAGE is reduced, and as a result, aggregation of fluorescent nanoparticle-labeled sRAGE on the test line is less likely to occur, and as a result, the fluorescence of the test spot is reduced. The presence of stimulating AGEs can be detected by the degree of inhibition of the fluorescence intensity of this test line. When an anti-G-BSA antibody is used in the test line, casein, PVA, or the like can be appropriately used because BSA cannot be used in the conjugate buffer or blocking buffer. As another example, a system in which an anti-OVA (ovalbumin) antibody is used for the test line and G-OVA is used as competitive AGEs can also be implemented.
　本開示のシステムまたはデバイスにおいて、好ましくは血球分離部を含み得る。血球分離部は試料として血球や血液成分が含まれるまたは含まれることが想定される場合に望ましい。血液には検出に阻害する成分があり得ることから、特に血球を分離しうることが有利であり得る。 The system or device of the present disclosure may preferably include a blood cell separator. The blood cell separator is desirable when the sample contains or is expected to contain blood cells or blood components. It may be particularly advantageous to be able to separate blood cells, as blood may have components that inhibit detection.
　さらなる局面において、本開示は、毛管現象による試料の展開を行うメンブレンを含む、生体マーカー分子の異常型を該生体マーカー分子とのコンジュゲート形成により検出または定量するためのデバイスまたはキットであって、該メンブレンが、生体マーカー分子の競合分子を含む検出部と、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部とを含み、該キットまたはデバイスは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含む、キットまたはデバイスを提供する。結合分子が試料中の生体マーカー分子に結合することにより、検出部における競合分子への結合が阻害され、蛍光強度の減少につながり得る。蛍光強度の減少に基づき、試料中の生体マーカー分子を定量することができる。別の局面において、本開示は、上記キットまたはデバイスにおいて使用するための、結合分子を標識する蛍光ナノ粒子、または蛍光ナノ粒子で標識された結合分子を含む組成物も提供し得る。検出部に競合分子を含む検出系の概要図を図１７に示す。 In a further aspect, the present disclosure is a device or kit for detecting or quantifying anomalous forms of a biomarker molecule by conjugate formation with the biomarker molecule, including a membrane that develops a sample by capillary phenomenon. A detection unit containing a competing molecule of a biomarker molecule, and a control unit containing a control binder that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having the ability to form a conjugate with the competing molecule. The kit or device comprises a sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane. When the binding molecule binds to the biomarker molecule in the sample, the binding to the competing molecule in the detection part is inhibited, which may lead to a decrease in fluorescence intensity. Biomarker molecules in the sample can be quantified based on the decrease in fluorescence intensity. In another aspect, the disclosure may also provide fluorescent nanoparticles that label the binding molecule, or compositions that include the binding molecule labeled with the fluorescent nanoparticles, for use in the kit or device. FIG. 17 shows a schematic diagram of a detection system containing a competing molecule in the detection unit.
　いくつかの実施形態において、生体マーカー分子はＡＧＥｓであり、生体マーカー分子の異常型は刺激性ＡＧＥｓであり、結合分子はｓＲＡＧＥであり、競合分子は、糖化処理なしＢＳＡ、糖化処理なしＯＶＡ、ＣＭＬ－ＢＳＡ、ＣＭＬ－ＯＶＡ、Ｇ－ＢＳＡもしくはＧ－ＯＶＡであり得る。特定の実施形態において、競合分子は、糖化処理なしＢＳＡ、糖化処理なしＯＶＡ、ＣＭＬ－ＢＳＡまたはＣＭＬ－ＯＶＡであり得、好ましくは、糖化処理なしＢＳＡまたはＣＭＬ－ＢＳＡであり得、最も好ましくは糖化処理なしＢＳＡまたはＣＭＬ－ＢＳＡであり得る。 In some embodiments, the biomarker molecule is AGEs, the variant of the biomarker molecule is stimulating AGEs, the binding molecule is sRAGE, and the competing molecules are BSA without glycation, OVA without glycation, CML. -Can be BSA, CML-OVA, G-BSA or G-OVA. In certain embodiments, the competing molecule can be unsaccharified BSA, unsaccharified OVA, CML-BSA or CML-OVA, preferably unsaccharified BSA or CML-BSA, most preferably saccharified. It can be untreated BSA or CML-BSA.
　（検出または定量方法）
　別の局面において、本開示は、生体マーカー分子の異常型を検出または定量するための方法であって、試料を提供する工程と、該試料と蛍光ナノ粒子で標識された結合分子とを混合する工程であって、該結合分子は生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する、工程と、本開示のデバイスまたはキットにおける前記メンブレンの前記試料接触部と、該混合された試料とを接触させる工程と、接触後、必要に応じて緩衝液を添加する工程とを含む、方法を提供する。 (Detection or quantification method)
In another aspect, the present disclosure is a method for detecting or quantifying anomalies of a biomarker molecule, in which the step of providing a sample is mixed with the bound molecule labeled with fluorescent nanoparticles. In the step, the binding molecule has the ability to form a conjugate with an atypical form of the biomarker molecule or a competing molecule thereof, and the step and the sample contact portion of the membrane in the device or kit of the present disclosure are mixed. Provided is a method including a step of contacting a sample and a step of adding a buffer solution as needed after the contact.
　別の局面において、本開示は、変性ＬＤＬまたは刺激性ＡＧＥｓを検出または定量するための方法であって、血液試料を提供する工程と、該血液試料と緩衝液および抗凝固剤（例えば、ヘパリン）とを混合する工程と、本開示のシステムまたはデバイスにおける試料接触部と、該混合された血液試料とを接触させる工程と、接触後、該緩衝液を添加する工程とを含む、方法を提供する。 In another aspect, the present disclosure is a method for detecting or quantifying denatured LDL or irritating AGEs, the step of providing a blood sample and the blood sample with a buffer and an anticoagulant (eg, heparin). Provided is a method comprising a step of mixing the blood sample, a step of bringing the sample contact portion in the system or device of the present disclosure into contact with the mixed blood sample, and a step of adding the buffer solution after the contact. ..
　いくつかの実施形態において、緩衝液としては、リン酸緩衝化生理食塩水（ＰＢＳ）などが挙げられるが、これらに限定されない。好ましい実施形態では、緩衝液は、ＰＢＳ（－）であり得る。緩衝液には、ウシ血清アルブミン（ＢＳＡ）等のアルブミン、カゼイン、ＰＶＡのような他の（その後の操作に影響のない）タンパク質が添加されても、されていなくてもよい。特定の実施形態において、緩衝液は、ＢＳＡ添加リン酸緩衝化生理食塩水（ＰＢＳ）（－）であり得る。 In some embodiments, the buffer solution includes, but is not limited to, phosphate buffered saline (PBS) and the like. In a preferred embodiment, the buffer can be PBS (−). The buffer may or may not contain albumin such as bovine serum albumin (BSA), casein, and other proteins (which do not affect subsequent manipulation) such as PVA. In certain embodiments, the buffer can be BSA-added phosphate buffered saline (PBS) (−).
　以上、本開示を、理解の容易のために好ましい実施形態を示して説明してきた。以下に、実施例に基づいて本開示を説明するが、上述の説明および以下の実施例は、例示の目的のみに提供され、本開示を限定する目的で提供したものではない。したがって、本開示の範囲は、本明細書に具体的に記載された実施形態にも実施例にも限定されず、特許請求の範囲によってのみ限定される。 The present disclosure has been described above by showing preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in the present specification, and is limited only by the scope of claims.
　（実施例１：カイコ中部絹糸腺抽出液からの一本鎖抗体の精製）
　標的配列ＵＳＡ下流にＢｉｏＥａｓｅ　ｔａｇ抗ＬＤＬ一本鎖抗体を挿入したＵＳＡ－Ｂｉｏｔｉｎ　ｔａｇ抗ＬＤＬ一本鎖抗体、標的配列ＵＳＡ下流にビオチンリガーゼ（ＢｉｒＡ）を挿入したＵＳＡ－ＢｉｒＡ、ならびにＧＡＬ４を中部絹糸腺特異的に発現させるセリシン１プロモーター（Ｓｅｒ１）－ＧＡＬの３つのコンストラクトを導入した遺伝子組換えカイコを作出した。このカイコは、中部絹糸腺特異的に、Ｂｉｏｔｉｎ　ｔａｇ抗ＬＤＬ一本鎖抗体およびＢｉｒＡを発現する。さらに、５令幼虫に１ｇ当たり２０μｇのビオチンを添加した餌を与え、抗ＬＤＬ一本鎖抗体のビオチン化に必要なビオチンを供給することによりビオチン化一本鎖抗体として生産することも可能である。本実施例においては、ビオチン化のされていない一本鎖抗体を使用した。繭を作る直前に中部絹糸腺絹糸腺を摘出し、ＴｒｉｔｏｎＸ－１００／ＰＢＳ（－）によりタンパク質を抽出した。 (Example 1: Purification of a single-chain antibody from a silk moth central silk gland extract)
USA-Biotin tag anti-LDL single-chain antibody with BioEase tag anti-LDL single-chain antibody inserted downstream of target sequence USA, USA-BirA with biotin ligase (BirA) inserted downstream of target sequence USA, and GAL4 in the central silk gland A recombinant silkworm into which three constructs of the sericin 1 promoter (Ser1) -GAL to be specifically expressed was introduced was produced. This silk moth expresses Biotin tag anti-LDL single-chain antibody and BirA specifically in the central silk gland. Furthermore, it is also possible to produce as a biotinylated single chain antibody by feeding 5th instar larvae with a diet supplemented with 20 μg of biotin per 1 g and supplying biotin necessary for biotinization of the anti-LDL single chain antibody. .. In this example, a non-biotinylated single chain antibody was used. Immediately before making the cocoon, the central silk gland was removed and the protein was extracted with Triton X-100 / PBS (-).
　凍結融解処理により中部絹糸腺抽出物からセリシンを除去した溶解物に、ＰＢＳで平衡化したＴＡＬＯＮ（Ｃｌｏｎｔｅｃｈ）レジン（Ｃｏを使用した金属キレートアフィニティクロマトグラフィレジン）を加え、終濃度が５ｍＭになるようイミダゾールを加えて２時間ゆっくりと撹拌した。レジンを１０ｍＭのイミダゾールを含むＰＢＳで洗浄した後、１Ｍのイミダゾールを含むＰＢＳで一本鎖抗体を溶出した。溶出画分を収集しＰＢＳ（－）で透析した。イムノクロマトアッセイには、透析後の試料を遠心式限外濾過フィルター（Ａｍｉｃｏｎ　Ｕｌｔｒａ，Ｍｅｒｋ）を用いて濃縮し、さらにバッファーを１０ｍＭのリン酸緩衝液（ｐＨ７．５）に置換したものを使用した。 TALON (Clontech) resin equilibrated with PBS (metal chelate affinity chromatography resin using Co) was added to the lysate from which sericin was removed from the central silk gland extract by freeze-thaw treatment, and imidazole was adjusted to a final concentration of 5 mM. Was added and the mixture was slowly stirred for 2 hours. The resin was washed with PBS containing 10 mM imidazole and then the single chain antibody was eluted with PBS containing 1 M imidazole. The eluted fraction was collected and dialyzed against PBS (-). For the immunochromatography assay, a sample after dialysis was concentrated using a centrifugal ultrafiltration filter (Amicon Ultra, Merc), and the buffer was further replaced with 10 mM phosphate buffer (pH 7.5).
　（結果）
　結果を図１に示す。示されるように、中部絹糸腺の発現系において、一本鎖抗体の発現に成功した。 (result)
The results are shown in FIG. As shown, the expression of the single chain antibody was successful in the expression system of the central silk gland.
　（実施例２：ラテラルフロー（イムノクロマト）アッセイ）
　（蛍光ナノ粒子標識ＣＴＬＤ１４の調製）
　酸化ＬＤＬ検出のためのイムノクロマトアッセイを行うため、酸化ＬＤＬを認識するＬＯＸ－１受容体のリガンド認識部位であるＣＴＬＤ１４を、シリカナノ粒子（ＱｕａｒｔｚＤｏｔ，古河ＡＥ）で修飾した。調製方法はＱｕａｒｔｚＤｏｔの取扱説明書に従って行ったが、概要は以下の通りである。 (Example 2: Lateral flow (immunochromatography) assay)
(Preparation of Fluorescent Nanoparticle Labeled CTLD14)
In order to perform an immunochromatographic assay for the detection of oxidized LDL, CTLD14, which is a ligand recognition site of the LOX-1 receptor that recognizes oxidized LDL, was modified with silica nanoparticles (QuartzDot, Furukawa AE). The preparation method was carried out according to the instruction manual of QuartzDot, and the outline is as follows.
　ＱｕａｒｔｚＤｏｔ（１００ｎｍ粒径）を、終濃度２ｍｇ／ｍｌになるようチューブ内でＮ－（４－アミノフェニル）マレイミド（終濃度２ｍｇ／ｍｌ）を含むジメチルホルムアミドと混合し３０℃で３０分間インキュベートした後、遠心分離し上清を除去した。チューブにＭＥＳ　ｂｕｆｆｅｒ（ｐＨ６．０，終濃度１００ｍＭ）、Ｎ－ヒドロキシコハク酸イミド（終濃度２３ｍｇ／ｍｌ）、塩酸－１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド（終濃度２．８８ｍｇ／ｍｌ）、ＣＴＬＤ１４溶液（終濃度３０μｇ／ｍｌ）を加えて超音波分散した後、３０℃で１時間インキュベートし、遠心分離し上清を除去した。沈殿にブロッキング剤Ａ、ＢとＭｉｌｌｉＱ水を１：２：７の割合で加え、超音波分散した後遠心分離し上清を除去した。沈殿を１０ｍＭのリン酸バッファー（ｐＨ７．５）で超音波分散した後遠心分離し上清を除去、再度リン酸バッファーを加えて超音波分散させ、蛍光ナノ粒子標識ＣＴＬＤ１４とした。 QuartzDot (100 nm particle size) is mixed with dimethylformamide containing N- (4-aminophenyl) maleimide (final concentration 2 mg / ml) in a tube to a final concentration of 2 mg / ml and incubated at 30 ° C. for 30 minutes. , Centrifuge and remove the supernatant. MES buffer (pH 6.0, final concentration 100 mM), N-hydroxysuccinimide (final concentration 23 mg / ml), hydrochloric acid-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (final concentration 2.88 mg) in a tube / Ml), CTLD14 solution (final concentration 30 μg / ml) was added and ultrasonically dispersed, then incubated at 30 ° C. for 1 hour, centrifuged and the supernatant was removed. Blocking agents A and B and MilliQ water were added to the precipitate at a ratio of 1: 2: 7, ultrasonically dispersed, and then centrifuged to remove the supernatant. The precipitate was ultrasonically dispersed with 10 mM phosphate buffer (pH 7.5), centrifuged to remove the supernatant, and the phosphate buffer was added again and ultrasonically dispersed to obtain a fluorescent nanoparticle-labeled CTLD14.
　（一本鎖抗体を用いたイムノクロマトストリップの調製）
　吸収パッドとメンブレンのみからなるハーフストリップのメンブレン上に、コントロールスポットとしてストレプトアビジン、テストスポットとして一本鎖抗体をそれぞれ０．５μｌスポットし、３７℃で１ｈ乾燥させた。スポットの乾燥後、室温で１％ＢＳＡ／ＰＢＳ溶液に浸して１５ｍｉｎゆっくり振盪し、ＭｉｌｌｉＱ水で軽く２回洗った後、さらに５ｍｉｎゆっくり振盪して洗浄した。洗浄後軽く水気を取り、キムワイプをひいたペーパータオル上で一晩風乾させた。検出には、蛍光検出器が必要になるが、簡易の検出器で十分対応可能である。本実施例では、蛍光イメージアナライザー：Ｔｙｐｈｏｏｎを使用した。 (Preparation of immunochromatographic strip using single chain antibody)
Streptavidin as a control spot and 0.5 μl of a single-chain antibody as a test spot were each spotted on a half-strip membrane consisting of an absorption pad and a membrane, and dried at 37 ° C. for 1 h. After the spot was dried, it was immersed in a 1% BSA / PBS solution at room temperature and gently shaken for 15 minutes, lightly washed twice with MilliQ water, and then gently shaken for another 5 minutes for washing. After washing, it was lightly drained and air-dried overnight on a paper towel with a Kimwipe. A fluorescence detector is required for detection, but a simple detector is sufficient. In this example, a fluorescence image analyzer: Typhoon was used.
　（結果）
　図２に示されるように、蛍光シリカナノ粒子標識ＣＴＬＤ１４を用いて、酸化ＬＤＬを検出することが可能であることが示された。図３は、各スポットの蛍光強度を測定した結果を示す。蛍光強度に基づき、酸化ＬＤＬ濃度を定量的に示すことが可能であることが明らかになった。 (result)
As shown in FIG. 2, it was shown that oxidized LDL can be detected using fluorescent silica nanoparticle labeled CTLD14. FIG. 3 shows the results of measuring the fluorescence intensity of each spot. It has been clarified that it is possible to quantitatively indicate the oxidized LDL concentration based on the fluorescence intensity.
　蛍光シリカナノ粒子による修飾は、塩を含まない緩衝液中で行う必要がある（ＰＢＳではなく、ＰＢ）。カイコＭＳＧ由来ＣＴＬＤ１４は非塩存在下でも機能を維持可能であったため蛍光シリカナノ粒子による修飾が可能であり、蛍光強度の測定により定量が可能なラテラルフローアッセイ系の開発が可能であった。 Modification with fluorescent silica nanoparticles must be performed in salt-free buffer (PB, not PBS). Since the silk moth MSG-derived CTLD14 was able to maintain its function even in the presence of non-salt, it could be modified with fluorescent silica nanoparticles, and it was possible to develop a lateral flow assay system that can be quantified by measuring the fluorescence intensity.
　（実施例３：イムノクロマトアッセイによる添加酸化ＬＤＬ検出）
　（材料および方法）
　テストスポットには、一本鎖抗体を塗布し、コントロールスポットには、ストレプトアビジンを塗布した。７５μｌのＰＢＳ（０．５％ＢＳＡを含む）に酸化ＬＤＬを添加し、モデル試料とした。２μｌの蛍光ナノ粒子標識ＣＴＬＤ１４をマイクロチューブ中のモデル試料に添加し混合した後、９６ｗｅｌｌマイクロプレートのｗｅｌｌ中に移し、ストリップを挿入して縦方向に展開した。約３０分後に展開を終え、ＴｙｐｈｏｏｎＦＬＡ９６００により蛍光画像を取得すると同時に、蛍光強度を測定した。 (Example 3: Detection of added oxidized LDL by immunochromatometry)
(Materials and methods)
A single chain antibody was applied to the test spot, and streptavidin was applied to the control spot. Oxidized LDL was added to 75 μl of PBS (containing 0.5% BSA) to prepare a model sample. After 2 μl of fluorescent nanoparticle labeled CTLD14 was added to the model sample in a microtube and mixed, it was transferred into the well of a 96-well microplate, stripped and vertically developed. The development was completed after about 30 minutes, and the fluorescence intensity was measured at the same time as the fluorescence image was acquired by TyphoonFLA9600.
　次に、試料として、１０００倍希釈、または、５０００倍希釈の血清に酸化ＬＤＬを添加したものを使用し、血清成分が展開を妨害するか検討した。 Next, as a sample, a 1000-fold diluted or 5000-fold diluted serum supplemented with oxidized LDL was used, and it was examined whether the serum components interfered with the development.
　（結果）
　結果を図３に示す。図３Ｂ，Ｃの上部の図が、蛍光画像を示し、下図が、蛍光強度（Ｃ：コントロールスポット、Ｔ：テストスポット）を示す。示されるように、展開後のストリップの各スポットの蛍光強度を測定したところ、酸化ＬＤＬ濃度を定量的に示すことが可能であることが明らかになった。血清試料を使用した結果を図４に示す。血清原液を対象にした場合、阻害がみられたが、１０００倍以上希釈した場合、検出に影響が無いことが確認された。 (result)
The results are shown in FIG. The upper part of FIGS. 3B and 3 shows the fluorescence image, and the lower figure shows the fluorescence intensity (C: control spot, T: test spot). As shown, when the fluorescence intensity of each spot of the unfolded strip was measured, it became clear that it was possible to quantitatively indicate the oxidized LDL concentration. The results of using the serum sample are shown in FIG. Inhibition was observed when the serum stock solution was used, but it was confirmed that there was no effect on detection when diluted 1000 times or more.
　（実施例４：高脂血症患者の血清を用いたイムノクロマトアッセイによる一本鎖抗体スポットの比較）
　図６の左図（動脈硬化性疾患予防ガイドラインに記載のある脂質異常症患者のＬＤＬ，　ＨＤＬ，　ＴＧの管理目標値と分類）を基に、ＬＤＬ，ＨＤＬ，　ＴＧの３者の値が、異常値、境界地、管理目標値のいずれにあるかにより、レベル０～４に分類した。さらに各分類と酸化ＬＤＬ値（国際公開第２０１６／０５１８０８号に準じて測定）との相関を解析した。次に、実施例２に従って、各レベルの血清中の酸化ＬＤＬを測定した。 (Example 4: Comparison of single chain antibody spots by immunochromatographic assay using serum of hyperlipidemic patients)
Based on the left figure of FIG. 6 (classified as the management target values of LDL, HDL, and TG of patients with dyslipidemia described in the guideline for prevention of arteriosclerosis), the values of LDL, HDL, and TG are abnormal. It was classified into levels 0 to 4 according to whether it was in the value, boundary area, or management target value. Furthermore, the correlation between each classification and the oxidized LDL value (measured according to International Publication No. 2016/051808) was analyzed. Next, the oxidized LDL in each level of serum was measured according to Example 2.
　（結果）
　結果を図７に示す。図７の中央図は、展開後の蛍光画像を示し、右図は蛍光強度を示す。レベル０の「血清１」に対して、異常と分類される項目がある検体群（血清２～６）は、スポットの蛍光強度が強かった。したがって、酸化ＬＤＬ濃度の簡易・迅速な定量の値により、管理状態を知ることができることが示唆された。 (result)
The results are shown in FIG. The central view of FIG. 7 shows the fluorescence image after development, and the right figure shows the fluorescence intensity. In the sample group (serum 2 to 6) having an item classified as abnormal with respect to level 0 "serum 1", the fluorescence intensity of the spot was strong. Therefore, it was suggested that the control state can be known from the simple and rapid quantification value of the oxidized LDL concentration.
　（実施例５：全血中の酸化ＬＤＬの検出）
　（材料および方法）
　全血の入ったマイクロチューブに酸化ＬＤＬ、蛍光ナノ粒子標識ＣＴＬＤ１４を添加し混合後、ミクロプレートのｗｅｌｌに移し、血球除去用フィルターを付けたフルストリップ（テストスポットに、濃度の異なる一本鎖抗体を、コントロールスポットにストレプトアビジンを塗布）を挿入し、縦方向に展開させた。 (Example 5: Detection of oxidized LDL in whole blood)
(Materials and methods)
Oxidized LDL and fluorescent nanoparticle-labeled CTLD14 are added to a microtube containing whole blood, mixed, transferred to a well of a microplate, and a full strip with a blood cell removal filter (a single chain antibody having a different concentration in a test spot). Was applied to the control spot with streptavidin) and developed in the vertical direction.
　（結果）
　図８の右側の図に示すように、展開後に取得した蛍光画像から酸化ＬＤＬの添加試料において、テストスポットの蛍光強度の顕著な増大が確認され、全血中の酸化ＬＤＬの簡易・迅速な検出・定量が可能なことが確認された。 (result)
As shown in the figure on the right side of FIG. 8, a remarkable increase in the fluorescence intensity of the test spot was confirmed in the sample to which oxidized LDL was added from the fluorescence image acquired after development, and simple and rapid detection of oxidized LDL in whole blood was confirmed.・ It was confirmed that quantification is possible.
　（実施例６：シリカナノ粒子を用いたＡＧＥ検出のための蛍光ナノ粒子標識ｓＲＡＧＥの調製）
　シリカナノ粒子（ＱｕａｒｔｚＤｏｔ，古河ＡＥ）を用いた蛍光ナノ粒子標識ｓＲＡＧＥの調製方法はＱｕａｒｔｚＤｏｔの取扱説明書に修正を加えて行った。概要は以下の通りである。 (Example 6: Preparation of fluorescent nanoparticle labeled sRAGE for AGE detection using silica nanoparticles)
The method for preparing the fluorescent nanoparticles labeled sRAGE using silica nanoparticles (QuartzDot, Furukawa AE) was carried out by modifying the instruction manual of QuartzDot. The outline is as follows.
　ＱｕａｒｔｚＤｏｔ（１００ｎｍ粒径）を，終濃度２ｍｇ／ｍｌになるようチューブ内でＮ－（４－アミノフェニル）マレイミド（終濃度２ｍｇ／ｍｌ）を含むジメチルホルムアミドと混合し３０℃で３０分間インキュベートした後、遠心分離し上清を除去した。チューブにＭＥＳ　ｂｕｆｆｅｒ（ｐＨ６．０，終濃度１００ｍＭ）、Ｎ－ヒドロキシコハク酸イミド（終濃度２３ｍｇ／ｍｌ）、塩酸－１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド（終濃度２．８８ｍｇ／ｍｌ）、ｓＲＡＧＥ溶液（終濃度３０μｇ／ｍｌ）を加えて超音波分散した後、３０℃で１時間インキュベートし、遠心分離し上清を除去した。沈殿をブロッキングする際、ブロッキング剤にはＱｕａｒｔｚＤｏｔ付属のブロッキング剤Ａ、ＢとＭｉｌｌｉＱ水を１：２：７の割合で加えたもの、または０．０５－１．０％ＰＶＡを含むリン酸バッファーまたはトリスバッファー、または０．０５－２．５％カゼインを含むリン酸バッファーまたはトリスバッファーを用いた。ブロッキング剤を加えて超音波分散した後遠心分離し上清を除去した。沈殿を１０ｍＭのリン酸バッファー（ｐＨ７．５）で超音波分散した後遠心分離し上清を除去、再度リン酸バッファーを加えて超音波分散させ、蛍光ナノ粒子標識ｓＲＡＧＥとした。 QuartzDot (100 nm particle size) is mixed with dimethylformamide containing N- (4-aminophenyl) maleimide (final concentration 2 mg / ml) in a tube to a final concentration of 2 mg / ml and incubated at 30 ° C. for 30 minutes. , Centrifuge and remove the supernatant. MES buffer (pH 6.0, final concentration 100 mM), N-hydroxysuccinimide (final concentration 23 mg / ml), hydrochloric acid-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (final concentration 2.88 mg) in a tube After adding / ml) and sRAGE solution (final concentration 30 μg / ml) and ultrasonically dispersing, the mixture was incubated at 30 ° C. for 1 hour, centrifuged, and the supernatant was removed. When blocking the precipitate, the blocking agent is a mixture of blocking agents A, B and MilliQ water attached to QuartzDot in a ratio of 1: 2: 7, or a phosphate buffer containing 0.05-1.0% PVA. Tris buffer, or phosphate buffer or Tris buffer containing 0.05-2.5% casein was used. A blocking agent was added, and the mixture was ultrasonically dispersed and then centrifuged to remove the supernatant. The precipitate was ultrasonically dispersed with 10 mM phosphate buffer (pH 7.5), centrifuged to remove the supernatant, and the phosphate buffer was added again and ultrasonically dispersed to obtain a fluorescent nanoparticle-labeled sRAGE.
　全血の入ったマイクロチューブに蛍光ナノ粒子標識ｓＲＡＧＥを添加し混合後、ミクロプレートのｗｅｌｌに移し、血球除去用フィルターを付けたフルストリップ（テストスポットへの抗体塗布は無し、コントロールスポットにストレプトアビジンを塗布）を挿入し、縦方向に展開させた。 Fluorescent nanoparticle-labeled sRAGE is added to a microtube containing whole blood, mixed, transferred to a well of a microplate, and a full strip with a blood cell removal filter (no antibody application to the test spot, streptavidin in the control spot). Was inserted) and developed in the vertical direction.
　（結果）
　結果を図９に示す。展開後に取得した蛍光画像から蛍光ナノ粒子標識ｓＲＡＧＥが全血の影響を受けずフルストリップ上で展開可能なことを確認された。 (result)
The results are shown in FIG. From the fluorescent image obtained after unfolding, it was confirmed that the fluorescent nanoparticle labeled sRAGE can be unfolded on a full strip without being affected by whole blood.
　（実施例７：蛍光ナノ粒子を用いたラテラルフロー検出法によるＡＧＥｓの検出）
　蛍光ナノ粒子を用いたＡＧＥ検出試薬（シリカナノ粒子標識ｓＲＡＧＥ）の調製
　蛍光ナノ粒子（ＱｕａｒｔｚＤｏｔ，古河電工アドバンストエンジニアリング）標識ｓＲＡＧＥ調製方法はＱｕａｒｔｚＤｏｔの取扱説明書に修正を加えて行った。概要は以下の通りである。 (Example 7: Detection of AGEs by lateral flow detection method using fluorescent nanoparticles)
Preparation of AGE Detection Reagent (Silica Nanoparticle Labeled sRAGE) Using Fluorescent Nanoparticles Fluorescent nanoparticles (QuartzDot, Furukawa Denko Advanced Engineering) Labeled sRAGE Preparation method was performed by modifying the instruction manual of QuartzDot. The outline is as follows.
　ＱｕａｒｔｚＤｏｔ（１００ｎｍ粒径）を，終濃度２ｍｇ／ｍｌになるようチューブ内でＮ－（４－アミノフェニル）マレイミド（終濃度２ｍｇ／ｍｌ）を含むジメチルホルムアミドと混合し３０℃で３０分間インキュベートした後、遠心分離し上清を除去した。チューブにＭＥＳ　ｂｕｆｆｅｒ（ｐＨ６．０，終濃度１００ｍＭ）、Ｎ－ヒドロキシコハク酸イミド（終濃度２３ｍｇ／ｍｌ）、塩酸－１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド（終濃度２．８８ｍｇ／ｍｌ）、ｓＲＡＧＥ溶液（終濃度３０～５０μｇ／ｍｌ）を加えて超音波分散した後、３０℃で１時間インキュベートし、遠心分離し上清を除去した。沈殿をブロッキングする際、ブロッキング剤にはＱｕａｒｔｚＤｏｔ付属のブロッキング剤Ａ、ＢとＭｉｌｌｉＱ水を１：２：７の割合で加えたもの、または０．１－０．５％ＰＶＡを含むリン酸バッファー、または０．５－１．０％カゼインを含むリン酸バッファーを用いた。ブロッキング剤を加えて超音波分散した後遠心分離し上清を除去した。沈殿を１０ｍＭのリン酸バッファー（ｐＨ７．５）で超音波分散した後遠心分離し上清を除去、再度リン酸バッファーを加えて超音波分散させ、蛍光ナノ粒子標識ｓＲＡＧＥとした。 QuartzDot (100 nm particle size) is mixed with dimethylformamide containing N- (4-aminophenyl) maleimide (final concentration 2 mg / ml) in a tube to a final concentration of 2 mg / ml and incubated at 30 ° C. for 30 minutes. , Centrifuge and remove the supernatant. MES buffer (pH 6.0, final concentration 100 mM), N-hydroxysuccinimide (final concentration 23 mg / ml), hydrochloric acid-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (final concentration 2.88 mg) in a tube / Ml), sRAGE solution (final concentration 30-50 μg / ml) was added and ultrasonically dispersed, then incubated at 30 ° C. for 1 hour, centrifuged and the supernatant was removed. When blocking the precipitate, the blocking agent was a mixture of blocking agents A and B attached to QuartzDot and MilliQ water in a ratio of 1: 2: 7, or a phosphate buffer containing 0.1-0.5% PVA. Alternatively, a phosphate buffer containing 0.5-1.0% casein was used. A blocking agent was added, and the mixture was ultrasonically dispersed and then centrifuged to remove the supernatant. The precipitate was ultrasonically dispersed with 10 mM phosphate buffer (pH 7.5), centrifuged to remove the supernatant, and the phosphate buffer was added again and ultrasonically dispersed to obtain a fluorescent nanoparticle-labeled sRAGE.
　ラテラルフロー用ストリップの調製
　吸収パッド、メンブレン、サンプルパッド（血球分離パッド）からなるフルストリップ、または吸収パッドとメンブレンのみからなるハーフストリップの各々のメンブレン上の適切な箇所に、コントロールスポット用試料（ストレプトアビジン）、テストスポット用試料（ＣＭＬ－ＡＧＥ－ＢＳＡ，抗ＡＧＥ抗体等）をそれぞれ０．５μｌスポットし、３７℃で３０ｍｉｎ～２ｈ乾燥させた。ブロッキングする場合はスポットの乾燥後、室温でブロッキングバッファー（０．５－１．０％ＢＳＡ，０－０．０５％のＴｗｅｅｎ－２０を含む０．０５－０．１％カゼイン／ＰＢＳ，０－０．０５％のＴｗｅｅｎ－２０を含む０．０５－０．１％ＰＶＡ／ＰＢＳ，０．０５－０．１％カゼイン／ＴＢＳ，０－０．１％カゼインを含む１×，１／２×，１／５×Ｐｉｅｒｃｅ　Ｐｒｏｔｅｉｎ－ｆｒｅｅ（ＰＢＳ／ＴＢＳ）Ｂｌｏｃｋｉｎｇ　ｂｕｆｆｅｒ等）に浸して１５ｍｉｎゆっくり振盪し、ＭｉｌｌｉＱ水で軽く２回洗った後、さらに５ｍｉｎゆっくり振盪して洗浄した。洗浄後軽く水気を取り、キムワイプをひいたペーパータオル上で一晩風乾させた。 Preparation of Lateral Flow Strips A sample for control spots (streptavidin) on each membrane, either a full strip consisting of an absorption pad, a membrane, a sample pad (blood cell separation pad), or a half strip consisting of an absorption pad and a membrane only. Avidin) and a test spot sample (CML-AGE-BSA, anti-AGE antibody, etc.) were each spotted in 0.5 μl and dried at 37 ° C. for 30 min to 2 hours. For blocking, after the spot has dried, 0.05-0.1% casein / PBS, 0-containing a blocking buffer (0.5-1.0% BSA, 0-0.05% Tween-20) at room temperature. 0.05-0.1% PVA / PBS containing 0.05% Tween-20, 0.05-0.1% casein / TBS, 1x, 1/2x containing 0-0.1% casein , 1/5 × Pierce Protein-free (PBS / TBS) Blocking buffer, etc.) and gently shaken for 15 minutes, lightly washed twice with MilliQ water, and then slowly shaken for another 5 minutes. After washing, it was lightly drained and air-dried overnight on a paper towel with a Kimwipe.
　ラテラルフロー検出法によるＡＧＥｓの検出
　６０μｌの反応バッファー（０．５－１．０％ＢＳＡ，０－０．０５％のＴｗｅｅｎ－２０を含む０．０５－０．１％カゼイン／ＰＢＳ，０－０．０５％のＴｗｅｅｎ－２０を含む０．０５－０．１％ＰＶＡ／ＰＢＳ，０．０５－０．１％カゼイン／ＴＢＳ，０－０．１％カゼインを含む１×，１／２×，１／５×Ｐｉｅｒｃｅ　Ｐｒｏｔｅｉｎ－ｆｒｅｅ（ＰＢＳ／ＴＢＳ）Ｂｌｏｃｋｉｎｇ　ｂｕｆｆｅｒ等）にＡＧＥ溶液（ＣＭＬ－ＡＧＥ－ＢＳＡ，Ｇｌｕｃｏｓｅ－ＡＧＥ－ＢＳＡ，Ｇｌｙｃｅｒａｌｄｅｈｙｄｅ－ＡＧＥ－ＢＳＡ，Ｇｌｙｃｏｌａｌｄｅｈｙｄｅ－ＡＧＥ－ＢＳＡ等）を１０ｎｇ～４μｇ添加し、モデルサンプルとして使用した。血清を含むサンプルの場合、凍結保存された血清を氷中で解凍後１，０００×ｇで１５ｍｉｎ遠心し、上清をＰＢＳで５００または１０００倍に希釈したものを６０μｌ使用した。サンプルに検出試薬（ＱｕａｒｔｚＤｏｔ標識ｓＲＡＧＥ）を加えて混合し、マイクロプレートウェルに混合溶液を移した後、調製したラテラルフロー用のハーフストリップを挿入した。２０分から３０分経過しウェル内の混合液がストリップにすべて吸収された後、Ｔｙｐｈｏｏｎ　ＦＬＡ　９５００を用いてテストスポット及びコントロールスポットの蛍光強度を確認した。全血入りのサンプルを用いる場合、５００倍の反応バッファーで希釈した血液７５μｌにＡＧＥ溶液を添加し、検出試薬（ＱｕａｒｔｚＤｏｔ標識ｓＲＡＧＥ）と混合した。マイクロプレートウェルに混合溶液を移し、サンプルパッド部分に血球分離パッドの付いたフルストリップを挿入した。５分から１０分経過しストリップが混合液を吸収し終わった頃、ウェルに反応バッファーを５０μｌ追加しさらに３０分程度経過しすべての溶液が吸収された後、Ｔｙｐｈｏｏｎ　ＦＬＡ　９５００を用いてテストスポット及びコントロールスポットの蛍光強度を確認した。 Detection of AGEs by Lateral Flow Detection Method 0.05-0.1% Casein / PBS, 0-0 with 60 μl reaction buffer (0.5-1.0% BSA, 0-0.05% Tween-20) 0.05-0.1% PVA / PBS containing 0.05% Tween-20, 0.05-0.1% casein / TBS, 1x, 1/2x, containing 0-0.1% casein, 1/5 x Pierce Protein-free (PBS / TBS) Blocking buffer, etc.) with AGE solution (CML-AGE-BSA, Glucose-AGE-BSA, Glyceraldehyde-AGE-BSA, Glycolaldehyde-AGE-BSA, etc.) It was added and used as a model sample. In the case of a sample containing serum, the cryopreserved serum was thawed in ice and centrifuged at 1,000 × g for 15 minutes, and 60 μl of the supernatant diluted 500 or 1000 times with PBS was used. The detection reagent (QuartzDot-labeled sRAGE) was added to the sample and mixed, the mixed solution was transferred to a microplate well, and then the prepared half-strip for lateral flow was inserted. After 20 to 30 minutes had passed and all the mixed solution in the well was absorbed by the strip, the fluorescence intensity of the test spot and the control spot was confirmed using Typhoon FLA 9500. When a sample containing whole blood was used, an AGE solution was added to 75 μl of blood diluted with a 500-fold reaction buffer and mixed with a detection reagent (QuartzDot-labeled sRAGE). The mixed solution was transferred to a microplate well, and a full strip with a blood cell separation pad was inserted into the sample pad portion. After 5 to 10 minutes, when the strip has absorbed the mixture, add 50 μl of reaction buffer to the wells, and after about 30 minutes, after all the solutions have been absorbed, test spots and controls using Typhoon FLA 9500. The fluorescence intensity of the spot was confirmed.
　モデルサンプル，ＡＧＥ／ＰＢＳ（－）
　テストスポットに抗ＡＧＥｓ抗体を塗布し、緩衝液に試料（糖化処理無しＢＳＡまたはグルコース糖化ＢＳＡ）を添加して展開した。蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥに結合したＡＧＥがテストラインへの結合し蛍光強度の増大（イメージアナライザーで画像取得）が確認された（図１１Ａ）。さらに、テストスポットにＣＭＬ糖化－ＢＳＡ（ＣＭＬ－ＢＳＡ、拮抗用ＡＧＥｓ）を塗布し、緩衝液に試料（糖化処理無しＢＳＡまたはＣＭＬ―BSA）を添加して展開した。蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥのテストラインへの結合がＡＧＥｓより阻害され、蛍光強度の減少（イメージアナライザーで画像取得）が確認された（図１１Ｂ）。テストスポットにＣＭＬ－ＢＳＡを塗布した検出系の概要を図１７に示す。 Model sample, AGE / PBS (-)
Anti-AGEs antibody was applied to the test spot, and a sample (BSA without saccharification treatment or BSA with glucose saccharification) was added to the buffer solution for development. It was confirmed that the AGE bound to the fluorescent nanoparticle-labeled biotinylated sRAGE was bound to the test line and the fluorescence intensity was increased (image acquisition with an image analyzer) (FIG. 11A). Further, CML saccharification-BSA (CML-BSA, antagonistic AGEs) was applied to the test spot, and a sample (BSA without saccharification treatment or CML-BSA) was added to the buffer solution for development. The binding of fluorescent nanoparticle-labeled biotinylated sRAGE to the test line was inhibited by AGEs, and a decrease in fluorescence intensity (image acquisition with an image analyzer) was confirmed (Fig. 11B). FIG. 17 shows an outline of the detection system in which CML-BSA is applied to the test spot.
　血清中のＡＧＥｓの検出
　テストスポットに抗ＡＧＥｓ抗体を塗布し、ヒト血清に試料（糖化処理無しＢＳＡまたはグルコース糖化ＢＳＡ）を添加して展開した。蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥに結合したＡＧＥがテストラインへの結合し蛍光強度の増大が確認された（図１２Ａ）。さらに、テストスポットにＣＭＬ－ＢＳＡ（拮抗用ＡＧＥｓ）を塗布し、緩衝液に試料（糖化処理無しＢＳＡまたはCML BSA）を添加して展開した。蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥのテストラインへの結合がＡＧＥｓより阻害され、ＡＧＥｓ濃度に依存した蛍光強度の減衰が確認された（図１２Ｂ）。 Detection of AGEs in Serum Anti-AGEs antibody was applied to the test spot, and a sample (BSA without glycation treatment or BSA with glucose glycation) was added to human serum for development. It was confirmed that the AGE bound to the fluorescent nanoparticle-labeled biotinylated sRAGE was bound to the test line and the fluorescence intensity was increased (Fig. 12A). Further, CML-BSA (antagonistic AGEs) was applied to the test spot, and a sample (BSA without saccharification treatment or CML BSA) was added to the buffer solution for development. Binding of fluorescent nanoparticle-labeled biotinylated sRAGE to the test line was inhibited by AGEs, and attenuation of fluorescence intensity depending on the concentration of AGEs was confirmed (Fig. 12B).
　全血中のＡＧＥｓの検出
　テストスポットに抗ＡＧＥｓ抗体を塗布し、ヒト新鮮血に試料（糖化処理無しＢＳＡまたはグルコース糖化ＢＳＡ）を添加して展開した。蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥに結合したＡＧＥがテストラインへの結合し蛍光強度の増大が確認された（図１３Ａ）。さらに、テストスポットにＣＭＬ－ＢＳＡ（拮抗用ＡＧＥｓ）を塗布し、ヒト新鮮血に試料（糖化処理無しＢＳＡまたはＣＭＬBSA）を添加して展開した。蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥのテストラインへの結合がＡＧＥｓより阻害され、蛍光強度の減少が確認された（図１３Ｂ）。 Detection of AGEs in whole blood Anti-AGEs antibody was applied to the test spot, and a sample (BSA without glycation treatment or BSA with glucose glycation) was added to fresh human blood for development. It was confirmed that the AGE bound to the fluorescent nanoparticle-labeled biotinylated sRAGE was bound to the test line and the fluorescence intensity was increased (FIG. 13A). Further, CML-BSA (antagonistic AGEs) was applied to the test spot, and a sample (BSA without saccharification treatment or CMLBSA) was added to fresh human blood for development. The binding of fluorescent nanoparticle-labeled biotinylated sRAGE to the test line was inhibited by AGEs, and a decrease in fluorescence intensity was confirmed (Fig. 13B).
　全血中のＡＧＥｓ検出（個人の体調不良を血液１滴で評価する系とした使用例）
　テストスポットにＣＭＬ－ＢＳＡ（拮抗用ＡＧＥｓ）を塗布し、健常者２名から、通常の時期、および、過労による体調不良が１週間程度継続した時期にランセットにより指から血液１滴を採血し、採血直後の新鮮血に試料（糖化処理無しＢＳＡ　または　ＣＭＬ―ＢＳＡ）を添加して展開し、蛍光強度の減少幅を比較したところ、体調不良時には蛍光強度の減少幅が低いことが確認された（図１４）。これは、体調不良時に蓄積が予想されるＡＧＥｓにより血中ＡＧＥｓ濃度が上昇し、添加したＡＧＥｓ（ＣＭＬ―ＢＳＡ）による阻害効果が見え難くなるためと思われる。 Detection of AGEs in whole blood (Example of use as a system for evaluating individual poor physical condition with one drop of blood)
CML-BSA (antagonistic AGEs) was applied to the test spot, and one drop of blood was collected from two healthy subjects by lancet during normal times and when poor physical condition due to overwork continued for about a week. When a sample (BSA without saccharification treatment or CML-BSA) was added to fresh blood immediately after blood collection and developed, and the decrease in fluorescence intensity was compared, it was confirmed that the decrease in fluorescence intensity was low when the patient was in poor physical condition (the decrease in fluorescence intensity was low). FIG. 14). It is considered that this is because the blood AGEs concentration increases due to the AGEs that are expected to accumulate when the patient is in poor physical condition, and the inhibitory effect of the added AGEs (CML-BSA) becomes difficult to see.
　ＮＡＳＨ患者血清中のＡＧＥｓの検出
　肥満や生活習慣病から発症する肝疾患、非アルコール性脂肪性肝疾患（ｎｏｎａｌｃｏｈｏｌｉｃ　ｆａｔｔｙ　ｌｉｖｅｒ　ｄｉｓｅａｓｅ：ＮＡＦＬＤ）は、わが国の肥満人口の増加を受け急増し、成人の２０～４０％が罹患する国民病になりつつある。ＮＡＦＬＤは、進行することのまれな非アルコール性脂肪肝（ｎｏｎａｌｃｏｈｏｌｉｃ　ｆａｔｔｙ　ｌｉｖｅｒ：ＮＡＦＬ）と肝硬変・肝細胞ガンへ進行する慢性進行性の非アルコール性脂肪肝炎（ｎｏｎａｌｃｏｈｏｌｉｃ　ｓｔｅａｔｏｈｅｐａｔｉｔｉｓ：ＮＡＳＨ）に分かれる。ＮＡＦＬＤから進行性のＮＡＳＨを判別することは極めて重要である。
　テストスポットに抗ＡＧＥｓ抗体を塗布し、ヒト血清を添加し展開した。血清中のＡＧＥが蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥに結合した結果、テストライン上に凝集し、蛍光強度の増大として、ＡＧＥｓ濃度を測定することができた（図１５Ａ）。さらに、テストスポットにＣＭＬ処理－ＢＳＡ（拮抗用ＡＧＥｓ）を塗布し、ヒト血清を添加して展開した。血清中のＡＧＥが蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥに結合した結果、テストライン上への凝集が阻害されるため、蛍光強度の減少としてＡＧＥｓ濃度を測定した（図１５Ｂ）。患者血清群で蛍光強度の減少が確認された。 Detection of AGEs in the serum of NASH patients The number of non-alcoholic fatty liver diseases (NAFLD), which is a liver disease caused by obesity and lifestyle diseases, has increased rapidly in response to the increase in the obese population in Japan, and is 20 adults. It is becoming a national disease that affects up to 40%. NAFLD is divided into nonalcoholic fatty liver (NAFL), which is rarely advanced, and chronic progressive nonalcoholic steatohepatitis (NASH), which progresses to cirrhosis and hepatocellular carcinoma. Determining progressive NASH from NAFLD is extremely important.
Anti-AGEs antibody was applied to the test spot, and human serum was added and developed. As a result of binding of AGE in serum to fluorescent nanoparticle-labeled biotinylated sRAGE, it aggregated on the test line, and the AGEs concentration could be measured as an increase in fluorescence intensity (FIG. 15A). Further, CML treatment-BSA (antagonistic AGEs) was applied to the test spot, and human serum was added and developed. As a result of binding of AGE in serum to fluorescent nanoparticle-labeled biotinylated sRAGE, aggregation on the test line was inhibited, so that the AGEs concentration was measured as a decrease in fluorescence intensity (FIG. 15B). A decrease in fluorescence intensity was confirmed in the patient serogroup.
　糖尿病合併症患者血清中のＡＧＥｓの検出
　テストスポットに抗ＡＧＥｓ抗体を塗布し、ヒト血清を添加して展開した。血清中のＡＧＥが蛍光ナノ粒子標識ビオチン化ｓＲＡＧＥに結合した結果、テストライン上に凝集し、蛍光強度の増大として、ＡＧＥｓ濃度を測定した（図１６）。ＨｂＡ１ｃの値の増大と蛍光強度に相関がみられた。 Detection of AGEs in the serum of diabetic complication patients Anti-AGEs antibody was applied to the test spot, and human serum was added and developed. As a result of binding of AGE in serum to fluorescent nanoparticle-labeled biotinylated sRAGE, it aggregated on the test line, and the AGEs concentration was measured as an increase in fluorescence intensity (FIG. 16). There was a correlation between the increase in HbA1c value and the fluorescence intensity.
　（注記）
　以上のように、本開示の好ましい実施形態を用いて本開示を例示してきたが、本開示は、この実施形態に限定して解釈されるべきものではない。本開示は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。当業者は、本開示の具体的な好ましい実施形態の記載から、本開示の記載および技術常識に基づいて等価な範囲を実施することができることが理解される。本明細書において引用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。本願は、２０１９年１１月８日に出願された日本国特許出願２０１９－２０３４４３号の優先権の利益を主張し、その内容が本明細書に対する参考として援用されるべきであることが理解される。 (Note)
As described above, the present disclosure has been illustrated using the preferred embodiments of the present disclosure, but the present disclosure should not be construed as being limited to this embodiment. It is understood that the present disclosure should be construed only by the claims. It will be understood from those skilled in the art that the description of the specific preferred embodiments of the present disclosure will enable equivalent scope to be implemented based on the description of the present disclosure and common general technical knowledge. The patents, patent applications and documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Understood. It is understood that the present application claims the priority benefit of Japanese Patent Application No. 2019-203443 filed on November 8, 2019, the contents of which should be incorporated as a reference to the present specification. ..
　本開示は、疾患の診断、又は予測診断を産業とする分野において有用である。 This disclosure is useful in the field of disease diagnosis or predictive diagnosis.
配列番号１：ＣＴＬＤ１４をコードする核酸配列
配列番号２：ＣＴＬＤ１４をコードするアミノ酸配列
配列番号３：ＲＡＧＥの核酸配列
配列番号４：ＲＡＧＥのアミノ核酸配列
配列番号５：本発明で使用されるｓＲＡＧＥの核酸配列
配列番号６：本発明で使用されるｓＲＡＧＥのアミノ核酸配列
配列番号７：本発明でしようされる一本鎖抗体のアミノ酸配列 SEQ ID NO: 1: Nucleic acid sequence encoding CTLD14 SEQ ID NO: 2: Amino acid sequence encoding CTLD14 SEQ ID NO: 3: RAGE nucleic acid sequence SEQ ID NO: 4: RAGE amino nucleic acid sequence SEQ ID NO: 5: sRAGE nucleic acid used in the present invention SEQ ID NO: 6: Amino acid sequence of sRAGE used in the present invention SEQ ID NO: 7: Amino acid sequence of the single-stranded antibody used in the present invention

Claims (26)

  1. 　毛管現象による試料の展開を行うメンブレンを含む、生体マーカー分子の異常型を該生体マーカー分子とのコンジュゲート形成により検出または定量するためのデバイスまたはキットであって、該メンブレンが、
    　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
    　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
    を含み、
    　該キットまたはデバイスは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含む、キットまたはデバイス。     A device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule, which comprises a membrane for developing a sample by capillarity, wherein the membrane is:
    A detection unit containing a detection binder that specifically binds to a biomarker molecule or a competing molecule thereof, and a control bond that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having the ability to form a conjugate with the competing molecule. Including the control part containing the agent,
    The kit or device comprises a sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
  2. 　前記メンブレンが、上流から下流にかけて、前記検出部、および前記コントロール部をこの順番で含む、請求項１に記載のキットまたはデバイス。 The kit or device according to claim 1, wherein the membrane includes the detection unit and the control unit in this order from upstream to downstream.
  3. 　前記試料接触部、前記検出部、および前記コントロール部が、相互に毛管現象で試料が浸透するように配置または連結されている、請求項１および１Ａに記載のキットまたはデバイス。 The kit or device according to claim 1 and 1A, wherein the sample contact portion, the detection portion, and the control portion are arranged or connected to each other so that the sample permeates through a capillary phenomenon.
  4. 前記生体マーカー分子はＬＤＬまたはＡＧＥｓである、請求項１～３のいずれか一項に記載のデバイスまたはキット。 The device or kit according to any one of claims 1 to 3, wherein the biological marker molecule is LDL or AGEs.
  5. 前記生体マーカー分子の異常型は変性ＬＤＬまたは刺激性ＡＧＥｓである、請求項１～４のいずれか一項に記載のデバイスまたはキット。 The device or kit according to any one of claims 1 to 4, wherein the aberrant form of the biomarker molecule is denatured LDL or stimulating AGEs.
  6. 前記結合分子はＣＴＬＤ１４またはｓＲＡＧＥである、請求項１～５のいずれか一項に記載のデバイスまたはキット。 The device or kit according to any one of claims 1 to 5, wherein the binding molecule is CTLD14 or sRAGE.
  7. 前記検出用結合剤は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメント、あるいは、抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントである、請求項１～６のいずれか一項に記載のデバイスまたはキット。 Any of claims 1 to 6, wherein the detection binding agent is an anti-LDL antibody, an anti-modified LDL antibody or an anti-ApoB antibody or an antigen-binding fragment thereof, or an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof. The device or kit described in one paragraph.
  8. 前記キットまたはデバイスは、前記生体マーカー分子の競合分子をさらに含む、請求項１～７のいずれか一項に記載のデバイスまたはキット。 The device or kit according to any one of claims 1 to 7, wherein the kit or device further comprises a competing molecule of the biomarker molecule.
  9. 前記生体マーカー分子はＬＤＬであり、前記生体マーカー分子の異常型は変性ＬＤＬであり、前記結合分子はＣＴＬＤ１４であり、前記検出用結合剤は抗ＬＤＬ抗体、抗変性ＬＤＬ抗体もしくは抗ＡｐｏＢ抗体またはそれらの抗原結合フラグメントである、請求項１～８のいずれか一項に記載のデバイスまたはキット。 The biomarker molecule is LDL, the variant of the biomarker molecule is denatured LDL, the binding molecule is CTLD14, and the detection binding agent is anti-LDL antibody, anti-modified LDL antibody or anti-ApoB antibody or them. The device or kit according to any one of claims 1 to 8, which is an antigen-binding fragment of the above.
  10. 前記生体マーカー分子はＡＧＥｓであり、前記生体マーカー分子の異常型は刺激性ＡＧＥｓであり、前記結合分子はｓＲＡＧＥであり、前記検出用結合剤は抗ＢＳＡ抗体もしくは抗ＯＶＡ抗体またはその抗原結合フラグメントであり、前記キットまたはデバイスは、前記生体マーカー分子の競合分子をさらに含み、該競合分子はＧ－ＢＳＡもしくはＧ－ＯＶＡである、請求項１～８のいずれか一項に記載のデバイスまたはキット。 The biomarker molecule is AGEs, the variant of the biomarker molecule is stimulating AGEs, the binding molecule is sRAGE, and the detection binding agent is an anti-BSA antibody or anti-OVA antibody or an antigen-binding fragment thereof. The device or kit according to any one of claims 1 to 8, wherein the kit or device further comprises a competing molecule of the biomarker molecule, wherein the competing molecule is G-BSA or G-OVA.
  11. 前記蛍光ナノ粒子は、検出試薬として提供される、請求項１～１０のいずれか一項に記載のデバイスまたはキット。 The device or kit according to any one of claims 1 to 10, wherein the fluorescent nanoparticles are provided as a detection reagent.
  12. 前記蛍光ナノ粒子は、前記メンブレン中で、試料混合物として提供される、請求項１～１０のいずれか一項に記載のデバイスまたはキット。 The device or kit according to any one of claims 1 to 10, wherein the fluorescent nanoparticles are provided as a sample mixture in the membrane.
  13. 前記試料接触部が、血球分離部を含む、請求項１～１２のいずれか一項に記載のデバイスまたはキット。 The device or kit according to any one of claims 1 to 12, wherein the sample contact portion includes a blood cell separation portion.
  14. 前記血球分離部が、ＦＵＳＩＯＮ５、ＬＦ１、ＭＦ１、およびＶＦ２から選択される、請求項１３に記載のデバイスまたはキット。 13. The device or kit of claim 13, wherein the blood cell separator is selected from FUSION5, LF1, MF1, and VF2.
  15. 前記ＣＴＬＤ１４が、ビオチン化、Ｈｉｓタグ付加、Ｍｙｃタグ付加、Ｆｌａｇタグ付加、Ｅタグ付加、またはＳｔｒｅｐタグ付加なされており、それぞれの場合において、前記コントロール用結合剤は、ストレプトアビジン、抗Ｈｉｓ抗体、抗Ｍｙｃ抗体、抗Ｆｌａｇ抗体、抗Ｅタグ抗体、またはＳｔｒｅｐ－Ｔａｃｔｉｎである、請求項４に記載のデバイスまたはキット。 The CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case, the control binder is streptavidin, an anti-His antibody, The device or kit of claim 4, which is an anti-Myc antibody, an anti-Flag antibody, an anti-E-tag antibody, or a Strept-Tactin.
  16. 前記ＣＴＬＤ１４が、カイコ型糖鎖を有する、請求項６に記載のデバイスまたはキット。 The device or kit according to claim 6, wherein the CTLD 14 has a silk moth-type sugar chain.
  17. 前記ＣＴＬＤ１４がビオチン化されており、前記コントロール用結合剤が、ストレプトアビジンである、請求項６に記載のデバイスまたはキット。 The device or kit of claim 6, wherein the CTLD14 is biotinylated and the control binder is streptavidin.
  18. 前記試料が、血液試料である、請求項１～１７のいずれか一項に記載のデバイスまたはキット。 The device or kit according to any one of claims 1 to 17, wherein the sample is a blood sample.
  19. 前記血液試料が、血清または全血である、請求項１８に記載のデバイスまたはキット。 The device or kit of claim 18, wherein the blood sample is serum or whole blood.
  20. 生体マーカー分子の異常型を検出または定量するための方法であって、
    　試料を提供する工程と、
    　該試料と蛍光ナノ粒子で標識された結合分子とを混合する工程であって、該結合分子は生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する、工程と、
    　請求項１～１８のいずれか一項に記載のデバイスまたはキットにおける前記メンブレンの前記試料接触部と、該混合された試料とを接触させる工程と、
    　接触後、必要に応じて緩衝液を添加する工程と
    を含む、方法。     A method for detecting or quantifying abnormal types of biological marker molecules.
    The process of providing the sample and
    A step of mixing the sample with a binding molecule labeled with fluorescent nanoparticles, wherein the binding molecule has the ability to form a conjugate with an aberrant form of a biomarker molecule or a competing molecule thereof.
    A step of contacting the sample contact portion of the membrane with the mixed sample in the device or kit according to any one of claims 1 to 18.
    A method comprising the step of adding a buffer as needed after contact.
  21. 　蛍光ナノ粒子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、
    　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
    　試料接触部と
    　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
    　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
    を含み、
    　該蛍光ナノ粒子は、該結合分子を標識することにより使用される、
    組成物。     A composition for use in a device, system or kit for detecting or quantifying anomalies of biomarker molecules, including fluorescent nanoparticles.
    The device, system or kit comprises a membrane that develops a sample by capillary action.
    The sample contact part and the detection part containing a detection binder that specifically binds to the biomarker molecule or its competing molecule and the abnormal type of the biomarker molecule or the binding molecule having the ability to form a conjugate with the competing molecule Includes a control unit containing a control binder
    The fluorescent nanoparticles are used by labeling the binding molecule.
    Composition.
  22. 　蛍光ナノ粒子で標識された結合分子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、該結合分子は、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有し、
    　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
    　試料接触部と
    　生体マーカー分子またはその競合分子に特異的に結合する検出用結合剤を含む検出部と
    　該結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
    を含む、
    組成物。     A composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising a binding molecule labeled with fluorescent nanoparticles, said binding molecule of the biomarker molecule. Has the ability to form conjugates with atypical or competing molecules thereof,
    The device, system or kit comprises a membrane that develops a sample by capillary action.
    It includes a sample contact part, a detection part containing a detection binder that specifically binds to a biological marker molecule or a competing molecule thereof, and a control part that contains a control binder that specifically binds to the binding molecule.
    Composition.
  23. 　毛管現象による試料の展開を行うメンブレンを含む、生体マーカー分子の異常型を該生体マーカー分子とのコンジュゲート形成により検出または定量するためのデバイスまたはキットであって、該メンブレンが、
    　生体マーカー分子の競合分子を含む検出部と
    　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
    を含み、
    　該キットまたはデバイスは、試料接触部および蛍光ナノ粒子をメンブレンの一部としてまたは別要素として含む、キットまたはデバイス。     A device or kit for detecting or quantifying an abnormal form of a biomarker molecule by conjugate formation with the biomarker molecule, which comprises a membrane for developing a sample by capillarity, wherein the membrane is:
    It includes a detection part containing a competing molecule of a biomarker molecule and a control part containing a control binder that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having a conjugate forming ability with the competing molecule.
    The kit or device comprises a sample contact and fluorescent nanoparticles as part of or as a separate element of the membrane.
  24. 　蛍光ナノ粒子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、
    　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
    　試料接触部と
    　生体マーカー分子の競合分子含む検出部と
    　生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
    を含み、
    　該蛍光ナノ粒子は、該結合分子を標識することにより使用される、
    組成物。     A composition for use in a device, system or kit for detecting or quantifying anomalies of biomarker molecules, including fluorescent nanoparticles.
    The device, system or kit comprises a membrane that develops a sample by capillary action.
    Includes a sample contact part, a detection part containing a competing molecule of a biomarker molecule, and a control part containing a control binder that specifically binds to an abnormal form of the biomarker molecule or a binding molecule having the ability to form a conjugate with the competing molecule. ,
    The fluorescent nanoparticles are used by labeling the binding molecule.
    Composition.
  25. 　蛍光ナノ粒子で標識された結合分子を含む、生体マーカー分子の異常型を検出または定量するためのデバイス、システムまたはキットにおいて使用するための組成物であって、該結合分子は、生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有し、
    　該デバイス、システムまたはキットは、毛管現象による試料の展開を行うメンブレンを含み、該メンブレンが、
    　試料接触部と
    　生体マーカー分子の競合分子を含む検出部と
    　該結合分子に特異的に結合するコントロール用結合剤を含むコントロール部と
    を含む、
    組成物。     A composition for use in a device, system or kit for detecting or quantifying anomalies of a biomarker molecule, comprising a binding molecule labeled with fluorescent nanoparticles, said binding molecule of the biomarker molecule. Has the ability to form conjugates with atypical or competing molecules thereof,
    The device, system or kit comprises a membrane that develops a sample by capillary action.
    It includes a sample contact part, a detection part containing a competing molecule of a biological marker molecule, and a control part containing a control binder that specifically binds to the binding molecule.
    Composition.
  26. 　生体マーカー分子の異常型を検出または定量するための方法であって、
    　試料を提供する工程と、
    　該試料と蛍光ナノ粒子で標識された結合分子とを混合する工程であって、該結合分子は生体マーカー分子の異常型またはその競合分子とコンジュゲート形成能を有する、工程と、
    　請求項２３に記載のデバイスまたはキットにおける前記メンブレンの前記試料接触部と、該混合された試料とを接触させる工程と、
    　接触後、必要に応じて緩衝液を添加する工程と
    を含む、方法。     A method for detecting or quantifying abnormal types of biological marker molecules.
    The process of providing the sample and
    A step of mixing the sample with a binding molecule labeled with fluorescent nanoparticles, wherein the binding molecule has the ability to form a conjugate with an aberrant form of a biomarker molecule or a competing molecule thereof.
    The step of contacting the sample contact portion of the membrane with the mixed sample in the device or kit according to claim 23.
    A method comprising the step of adding a buffer as needed after contact.
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