Simple and rapid quantitative method for denatured LDL and stimulatory AGEs

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

[Technical Field]

[0001] The present invention relates to a technique for easily and quickly quantitatively detecting modified LDL or irritating AGEs. [Background technology]

[0002] Oxidized LDL (so-called "bad cholesterol") is known as a risk factor for dyslipidemia, ischemic heart disease, and other conditions. Oxidized LDL is a collective term for LDL molecules that have undergone oxidative modification, and is a heterogeneous group of molecules with varying degrees of modification. The inventors have successfully developed a method for comprehensively detecting only oxidized LDL (the true bad cholesterol), which triggers disease in the body, from among oxidatively modified LDL molecules (Patent Document 1). However, the developed method requires specialized techniques and equipment and is intended for use in research and testing laboratories. Meanwhile, there is a need for a method for easily and quickly quantitatively evaluating the effects of lifestyle improvements, such as exercise and dietary habits, on the prevention of disease progression, and the status of disease management through medication and other measures.

[0003] AGEs (Advanced Glycation End Products) are the glycation products of proteins, and are a general term for a variety of structures. In recent years, it has been discovered that AGEs produced by glycative stress in the human body also exist, including irritant AGEs that can induce diabetic complications and age-related diseases (e.g., rheumatoid arthritis and Alzheimer's disease). The inventors have successfully developed a method for detecting disease-triggering AGEs from among the diverse structures of AGEs. However, like the oxidized LDL detection method, the developed method is intended for use in research and testing institutions. Therefore, there is a need for a simple and rapid method for quantitatively evaluating the effects of lifestyle improvements, such as exercise and dietary habits, on the prevention of disease progression, and the status of disease management through medication, etc. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 051808 Summary of the Invention [Means for solving the problem]

[0005] The present inventors have developed a technique for quantifying oxidized LDL or irritating AGEs, such as a lateral flow assay (immunochromatography). Specifically, the present quantitative technique uses a recognition element modified with fluorescent nanoparticles that recognize oxidized LDL or irritating AGEs, thereby achieving simple and rapid quantification of oxidized LDL or irritating AGEs.

[0006] 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 forming a conjugate with the biomarker molecule, comprising a membrane that develops a sample by capillary action, the membrane comprising: a detection unit containing a detection binding agent that specifically binds to a biomarker molecule or its competitor molecule; a control section containing a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with an abnormal form of the biomarker molecule or its competitor molecule; Including, The kit or device includes a sample contact portion and fluorescent nanoparticles as part of a membrane or as separate elements. (Item 2) Item 2. The kit or device according to Item 1, wherein the membrane comprises, from upstream to downstream, the detection section and the control section, in this order. (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 so that the sample penetrates into each other by capillary action. (Item 4) 4. The device or kit according to any one of items 1 to 3, wherein the biomarker molecule is LDL or AGEs. (Item 5) 5. The device or kit according to any one of items 1 to 4, wherein the abnormal form of the biomarker molecule is modified LDL or irritating AGEs. (Item 6) 6. The device or kit according to any one of items 1 to 5, wherein the binding molecule is CTLD14 or sRAGE. (Item 7) 7. The device or kit according to any one of items 1 to 6, wherein the detection binding agent is an anti-LDL antibody, an anti-denatured LDL antibody, an anti-ApoB antibody, or an antigen-binding fragment thereof, or an anti-BSA antibody, an anti-OVA antibody, or an antigen-binding fragment thereof. (Item 8) 8. The device or kit according to any one of items 1 to 7, further comprising a competitor molecule for the biomarker molecule. (Item 9) 9. The device or kit according to any one of items 1 to 8, wherein the biomarker molecule is LDL, the abnormal form of the biomarker molecule is denatured LDL, the binding molecule is CTLD14, and the detection binding agent is an anti-LDL antibody, an anti-denatured LDL antibody, or an anti-ApoB antibody, or an antigen-binding fragment thereof. (Item 10) The device or kit according to any one of Items 1 to 8, wherein the biomarker molecule is an AGE, the abnormal form of the biomarker molecule is an stimulatory AGE, the binding molecule is sRAGE, the detection binding agent is an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and the kit or device further comprises a competitor molecule of the biomarker molecule, wherein the competitor molecule is G-BSA or G-OVA. (Item 11) 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) 11. 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) 13. The device or kit according to any one of items 1 to 12, wherein the sample contact section includes a blood cell separation section. (Item 14) Item 14. The device or kit according to item 13, wherein the blood cell separation unit is selected from FUSION5, LF1, MF1, and VF2. (Item 15) 5. The device or kit of item 4, wherein the CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case the control binding agent is streptavidin, an anti-His antibody, an anti-Myc antibody, an anti-Flag antibody, an anti-E tag antibody, or Strep-Tactin. (Item 16) 7. The device or kit according to item 6, wherein the CTLD14 has a silkworm-type glycan. (Item 17) 7. The device or kit of item 6, wherein the CTLD14 is biotinylated and the control binding agent is streptavidin. (Item 18) 18. The device or kit according to any one of items 1 to 17, wherein the sample is a blood sample. (Item 19) 19. The device or kit according to item 18, wherein the blood sample is serum or whole blood. (Item 20) 1. A method for detecting or quantifying an aberrant form of a biomarker molecule, comprising: providing a sample; mixing the sample with a binding molecule labeled with a fluorescent nanoparticle, the binding molecule capable of forming a conjugate with an aberrant form of a biomarker molecule or a competitor thereof; A step of contacting the mixed sample with the sample contact portion of the membrane in the device or kit according to any one of items 1 to 18; After contacting, adding a buffer solution as needed; A method comprising: (Item 21) 1. A composition for use in a device, system or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising fluorescent nanoparticles, The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: Sample contact area and a detection unit containing a detection binding agent that specifically binds to a biomarker molecule or its competitor molecule; a control section containing a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with an abnormal form of the biomarker molecule or its competitor molecule; Including, The fluorescent nanoparticles are used by labeling the binding molecules. composition. (Item 22) 1. A composition for use in a device, system, or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising a binding molecule labeled with a fluorescent nanoparticle, the binding molecule being capable of forming a conjugate with the aberrant form of the biomarker molecule or a competitor molecule thereof; The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: Sample contact area and a detection unit containing a detection binding agent that specifically binds to a biomarker molecule or its competitor molecule; a control section containing a control binding agent that specifically binds to the binding molecule; Including, composition. (Item 23) A device or kit for detecting or quantifying an abnormal form of a biomarker molecule by forming a conjugate with the biomarker molecule, comprising a membrane that develops a sample by capillary action, the membrane comprising: a detection section containing a competitor molecule for the biomarker molecule; a control section containing a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with an abnormal form of the biomarker molecule or its competitor molecule; Including, The kit or device includes a sample contact portion and fluorescent nanoparticles as part of a membrane or as separate elements. (Item 24) 24. The kit or device of claim 23, having one or more of the features described above. (Item 25) 1. A composition for use in a device, system or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising fluorescent nanoparticles, The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: Sample contact area and a detection section containing a competitor molecule for the biomarker molecule; a control section containing a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with an abnormal form of the biomarker molecule or its competitor molecule; Including, The fluorescent nanoparticles are used by labeling the binding molecules. composition. (Item 26) 1. A composition for use in a device, system, or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising a binding molecule labeled with a fluorescent nanoparticle, the binding molecule being capable of forming a conjugate with the aberrant form of the biomarker molecule or a competitor molecule thereof; The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: Sample contact area and a detection section containing a competitor molecule for the biomarker molecule; a control section containing a control binding agent that specifically binds to the binding molecule; Including, composition. (Item 27) 27. The composition of claim 25 or 26, characterized by one or more of the above items. (Item 28) 1. A method for detecting or quantifying an aberrant form of a biomarker molecule, comprising: providing a sample; mixing the sample with a binding molecule labeled with a fluorescent nanoparticle, the binding molecule capable of forming a conjugate with an aberrant form of a biomarker molecule or a competitor thereof; Contacting the mixed sample with the sample contact portion of the membrane in the device or kit according to Item 23; After contacting, adding a buffer solution as needed; A method comprising: (Item 29) 29. The method of claim 28, having one or more of the features described above.

[0007] 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 forming a conjugate with the biomarker molecule, comprising a membrane that develops a sample by capillary action, the membrane comprising: Sample contact area and a detection unit containing a detection binding agent that specifically binds to a biomarker molecule or its competitor molecule; a control section containing a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with an abnormal form of the biomarker molecule or its competitor molecule; Including, The kit or device includes fluorescent nanoparticles as part of the membrane or as a separate element. (Item 2A) The device or kit according to item 1A, wherein the biomarker molecule is LDL or AGEs. (Item 3A) The device or kit according to item 1A, wherein the abnormal form of the biomarker molecule is modified LDL or irritating AGEs. (Item 4A) The device or kit of any one of items 1A to 3A, wherein the binding molecule is CTLD14 or sRAGE. (Item 5A) The device or kit of any one of Items 1A to 4A, wherein the detectable binding agent is an anti-LDL antibody, an anti-denatured LDL antibody, 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. (Item 6A) The device or kit according to any one of Items 1A to 5A, wherein the kit or device further comprises a competitor molecule for the biomarker molecule. (Item 7A) The device or kit of any one of Items 1A to 6A, wherein the biomarker molecule is LDL, the abnormal form of the biomarker molecule is denatured LDL, the binding molecule is CTLD14, and the detection binding agent is an anti-LDL antibody, an anti-denatured LDL antibody, or an anti-ApoB antibody, or an antigen-binding fragment thereof. (Item 8A) The device or kit according to any one of Items 1A to 6A, wherein the biomarker molecule is an AGE, the abnormal form of the biomarker molecule is a stimulatory AGE, the binding molecule is sRAGE, the detection binding agent is an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and the kit or device further comprises a competitor molecule of the biomarker molecule, wherein the competitor 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 separation section. (Item 12A) The device or kit according to Item 11A, wherein the blood cell separation unit is selected from FUSION5, LF1, MF1, and VF2. (Item 13A) 4B. The device or kit of item 4A, wherein the CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case the control binding agent is streptavidin, an anti-His antibody, an anti-Myc antibody, an anti-Flag antibody, an anti-E tag antibody, or Strep-Tactin. (Item 14A) The device or kit according to Item 4A, wherein the CTLD14 has a silkworm-type glycan. (Item 15A) The device or kit of item 4A, wherein the CTLD14 is biotinylated and the control binding agent 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) 1. A method for detecting or quantifying an aberrant form of a biomarker molecule, comprising: providing a sample; mixing the sample with a binding molecule labeled with a fluorescent nanoparticle, the binding molecule capable of forming a conjugate with an aberrant form of a biomarker molecule or a competitor thereof; contacting the mixed sample with the sample contact portion of the membrane in the device or kit according to any one of items 1A to 16A; After contacting, adding a buffer solution as needed; A method comprising: (Item 18A) 1. A composition for use in a device, system or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising fluorescent nanoparticles, The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: Sample contact area and a detection unit containing a detection binding agent that specifically binds to a biomarker molecule or its competitor molecule; a control section containing a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with an abnormal form of the biomarker molecule or its competitor molecule; Including, The fluorescent nanoparticles are used by labeling the binding molecules. composition. (Item 19A) 1. A composition for use in a device, system, or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising a binding molecule labeled with a fluorescent nanoparticle, the binding molecule being capable of forming a conjugate with the aberrant form of the biomarker molecule or a competitor molecule thereof; The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: Sample contact area and a detection unit containing a detection binding agent that specifically binds to a biomarker molecule or its competitor molecule; a control section containing a control binding agent that specifically binds to the binding molecule; A composition comprising:

[0008] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0009] The present disclosure provides a technique for easily and quickly quantifying disease-related oxidized LDL or irritating AGEs. Accordingly, the present disclosure provides a kit and method for detecting oxidized LDL or irritating AGEs, which are useful for diagnosing diseases (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease), evaluating the effectiveness of treatments, and taking preventive measures, as well as a substrate that can be used therefor. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows an overview of single chain antibody production. [Figure 2] FIG. 2 shows the results of detection of oxidized LDL by lateral flow (immunochromatography) assay. [Figure 3] FIG. 3 shows the results of measuring the fluorescence intensity of each spot on the strip in FIG. [Figure 4] FIG. 4 shows the results of detecting added oxidized LDL in serum by immunochromatographic assay. [Figure 5] FIG. 5 shows a schematic diagram of the principle of detection of modified LDL by lateral flow assay. [Figure 6] Figure 6 shows the correlation between oxidized LDL concentrations in the serum of hyperlipidemic patients and the management status of dyslipidemic patients as determined from their LDL, HDL, and TG concentrations. Based on the classification (left) of dyslipidemic patients based on whether their LDL, HDL, and TG values ​​were within the management target range or were determined to be abnormal, patient serum was classified into levels 0 to 4 (lower right) based on the number of LDL, HDL, and TG values ​​classified as abnormal, and the range of oxidized LDL concentrations for each level was organized (upper right). [Figure 7] FIG. 7 shows a comparison of oxidized LDL concentrations by immunochromatographic assay using sera from hyperlipidemic patients classified in FIG. [Figure 8] FIG. 8 shows the results of immunochromatographic assay for the detection of oxidized LDL added to whole blood samples. [Figure 9]Figure 9 shows the results of confirming whether fluorescent nanoparticle-labeled sRAGE can be developed normally on immunochromatography when whole blood is added. [Figure 10] FIG. 10 shows a schematic diagram of the principle of detection of irritant AGEs by lateral flow assay. [Figure 11] FIG. 11 shows the results of a lateral flow assay using a model sample. [Figure 12] Figure 12 shows the results of detecting AGEs in serum by lateral flow assay. The image on the left in Figure 12 shows an image acquired with an image analyzer, and the graph on the right shows the fluorescence intensity of each spot. [Figure 13] Figure 13 shows the results of detecting AGEs in whole blood by lateral flow assay. The image on the left in Figure 13 shows an image acquired with an image analyzer, and the graph on the right shows the fluorescence intensity of each spot. [Figure 14] Figure 14 shows the results of detecting AGEs in whole blood by lateral flow assay. The image on the left in Figure 14 shows an image acquired with an image analyzer, and the graph on the right shows the fluorescence intensity of each spot. [Figure 15] Figure 15 shows the results of detecting AGEs in the serum of NASH patients by lateral flow assay. The image on the left in Figure 15 shows an image acquired with an image analyzer, and the graph on the right shows the fluorescence intensity of each spot. [Figure 16] Figure 16 shows the results of lateral flow assay detection of AGEs in the serum of patients with diabetic complications. The image on the left in Figure 16 shows an image acquired with an image analyzer, and the graph on the right shows the fluorescence intensity of each spot. [Figure 17] FIG. 17 shows a schematic diagram of a detection system in which a competitor molecule (CML glycated BSA) is applied to the detection portion (test spot). DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure will be described below. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the relevant field unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will prevail.

[0012] (Definition of terms) The following are definitions of terms particularly used in this specification.

[0013] As used herein, "about" means ±10% of the indicated value.

[0014] As used herein, the term "system" refers to any system for performing detection, predictive diagnosis, pre-diagnosis, diagnosis, etc., and generally refers to a system that satisfies three conditions: it consists of one or more components, and if there are multiple components, those components interact and interact with each other, and exhibit harmonious behavior and function as a whole. A system can take any form, such as an apparatus, device, composition, or diagnostic agent. Therefore, systems are understood to encompass, for example, large-scale systems equipped with measurement devices, systems equipped with chromatography, kits and combinations utilizing immune reactions, and compositions containing antibodies (i.e., diagnostic agents that are in vitro pharmaceuticals containing monoclonal antibodies of markers), etc.

[0015] As used herein, the term "device" refers to any apparatus for performing detection, predictive diagnosis, pre-diagnosis, diagnosis, etc., and is composed of one or more components, and when there are multiple components, these elements are usually connected to each other. It is used to refer to any apparatus, instrument, tool, or thing used for a specific purpose, and is not limited to those that have mechanical or electrical functions. It usually includes at least one element operably linked to each other to enable a purpose (e.g., inspection, detection, diagnosis, etc.).

[0016] As used herein, a "kit" refers to a set of components to be provided, usually divided into two or more compartments. A kit refers to a unit in which a part (e.g., a membrane, a device, a reagent, etc.) is provided. When multiple reagents or devices are provided independently, it may be convenient to provide them as a kit. Such a kit preferably advantageously includes instructions or manuals describing how to use the provided part (e.g., a membrane or a device) or how to use the reagent.

[0017] As used herein, "instructions" refers to instructions to the user on how to use the present disclosure. The instructions contain language instructing on how to use the present disclosure. If necessary, the instructions are prepared in accordance with a format specified by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, the Food and Drug Administration (FDA) or the Department of Agriculture (USDA) in the United States), and it is clearly stated that the instructions have been approved by the regulatory agency. The instructions may be provided in paper form, but are not limited thereto, and may also be provided in the form of electronic media (e.g., an internet-provided homepage, email, social media, simple messages, etc.).

[0018] As used herein, the term "fluorescent nanoparticles" refers to nano-sized particles that can emit fluorescence of sufficient intensity to detect a target biological substance. Quantum dots (semiconductor nanoparticles) and fluorescent substance-holding nanoparticles are preferably used as fluorescent nanoparticles.

[0019] Quantum dots are semiconductor nanoparticles containing II-VI compounds, III-V compounds, or IV elements. Examples include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge. Phosphor-holding nanoparticles are nano-sized particles with a structure that uses an organic or inorganic particle as a host body and has multiple fluorescent substances (e.g., the quantum dots and fluorescent dyes) encapsulated within and / or adsorbed to its surface.

[0020] The fluorescent substance-having nanoparticles are preferably those in which the host and fluorescent substance have substituents or moieties with opposite charges, thereby causing electrostatic interaction. Examples of the fluorescent substance-having nanoparticles that can be used include quantum dot-having nanoparticles and fluorescent dye-having nanoparticles.

[0021] Examples of organic materials in the matrix include resins generally classified as thermosetting resins, such as melamine resin, urea resin, aniline resin, guanamine resin, phenolic resin, xylene resin, and furan resin; resins generally classified as thermoplastic resins, such as styrene resin, acrylic resin, acrylonitrile resin, AS resin (acrylonitrile-styrene copolymer), and ASA resin (acrylonitrile-styrene-methyl acrylate copolymer); other resins such as polylactic acid; and polysaccharides. Examples of inorganic materials in the matrix include silica and glass.

[0022] Quantum dot-assembled nanoparticles Quantum dot-assembled nanoparticles have a structure in which the quantum dots are encapsulated in the host material and / or adsorbed to its surface. When the quantum dots are encapsulated in the host material, the quantum dots are simply dispersed within the host material, and may or may not be chemically bonded to the host material itself.

[0023] Fluorescent dye-conjugated nanoparticles Fluorescent dye-holding nanoparticles have a structure in which a fluorescent dye is encapsulated within the matrix and / or adsorbed to its surface. Examples of fluorescent dyes 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 pyrromethene-based dye molecules. Examples of fluorescent dyes that can be used include Alexa Fluor (registered trademark, manufactured by Invitrogen) dye molecules, BODIPY (registered trademark, manufactured by Invitrogen) dye molecules, Cy (registered trademark, manufactured by GE Healthcare) dye molecules, HiLyte (registered trademark, manufactured by Anaspec) dye molecules, DyLight (registered trademark, manufactured by Thermo Scientific) dye molecules, ATTO (registered trademark, manufactured by ATTO-TEC) dye molecules, MFP (registered trademark, manufactured by Mobitec) dye molecules, CF (registered trademark, manufactured by Biotium) dye molecules, DY (registered trademark, manufactured by DYOMICS) dye molecules, and CAL (registered trademark, manufactured by BioSearch Technologies) dye molecules.

[0024] 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, tetramethylrhodamine, X-rhodamine, sulforhodamine B, sulforhodamine 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 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 (all manufactured by Invitrogen), methoxycoumarin, coumarin 6, coumarin 7, sulfocoumarin 6, sulfocoumarin 7, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 594, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750 (registered trademark, manufactured by Anaspec), DyLight 350, DyLight 405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight650, DyLight 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 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, ATTO740 (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, manufactured by Biotium), 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 (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 615, CAL Fluor Red 635, Pulsar 650 (Registered Trademark, BioSearchTechnologies), 5,10,15,20-tetraphenylporphine tetrasulfonic acid, zinc 5,10,15,20-tetraphenylporphine tetrasulfonic acid, phthalocyanine tetrasulfonic acid, zinc phthalocyanine tetrasulfonic acid, 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, Examples of fluorescent dyes that can be used include 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-. These fluorescent dyes may be used alone or in combination. The generic names for these dye molecules are based on the main structure (skeleton) of the compound or its registered trademark, and those skilled in the art can adequately grasp the scope of the fluorescent dyes that belong to each group without excessive trial and error. When a fluorescent dye is encapsulated in a matrix, it is sufficient that the fluorescent dye is dispersed within the matrix, and it may or may not be chemically bound to the matrix itself.

[0025] As used herein, the term "biomarker molecule" refers to a substance that serves as an indicator for tracking whether a person has or is at risk of a certain condition (e.g., a disease, a disorder, etc.). Examples of such markers include genes, gene products, metabolites, enzymes, etc. In the present invention, biomarker molecules include LDL, AGEs, or analogs thereof. Some biomarker molecules have different levels or forms between healthy and non-healthy states, and those that are characteristically found in non-healthy states are particularly referred to herein as "abnormal biomarker molecules." Abnormal biomarker molecules are particularly closely associated with disease and can therefore be detected in the present invention. Abnormal biomarker molecules are often modified forms of biomarkers that are not normally observed in healthy individuals, or are observed in low amounts, if at all. Examples of abnormal biomarker molecules include modified LDL and irritating AGEs.

[0026] As used herein, the term "competitor molecule" refers to a molecule that binds to a target in competition with a binding molecule that binds to the target. Since a competitor molecule competes with a binding molecule for binding to a target, the presence of the competitor molecule reduces the amount of binding of the binding molecule. Therefore, the use of the competitor molecule makes it possible to indirectly measure the amount of the binding molecule or binding (e.g., conjugate formation).

[0027] As used herein, the term "conjugate" refers to the binding of a certain object to another entity to form a single entity, and this ability is referred to as "conjugate-forming ability." For example, CLTD14 is an example of such a molecule for denatured LDL, and sRAGE is an example of such a molecule for irritant AGEs. Those skilled in the art can identify other molecules as appropriate.

[0028] As used herein, the term "detection binding agent" refers to a molecule (e.g., an anti-LDL antibody, an anti-denatured LDL antibody, 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) that specifically binds to the biomarker molecule to be detected or an abnormal form thereof, or a competitor molecule thereof, in the detection portion of the membrane (e.g., a test spot or line) in the device or kit of the present disclosure.

[0029] As used herein, a "control binding agent" refers to a molecule that specifically binds to a binding molecule capable of forming a conjugate with an aberrant form of a biomarker molecule or a binding molecule capable of forming a conjugate with a competitor molecule of an aberrant form of a biomarker molecule in a control portion of a membrane (e.g., a control spot or line) in a device or kit of the present disclosure. In one embodiment, the control binding agent can be a binding molecule (e.g., CTLD14) capable of forming a conjugate with a molecule that specifically binds to an abnormal form of a biomarker molecule (e.g., denatured LDL), or a binding molecule (sRAGE) capable of forming a conjugate with a competing molecule (e.g., G-BSA) that competes with the biomarker molecule (e.g., stimulatory AGEs). Once the abnormal form of a biomarker molecule is identified, one skilled in the art can, as appropriate, identify or generate a molecule that specifically binds to the binding molecule capable of forming a conjugate with that molecule (e.g., CTLD14 in the case of denatured LDL) (e.g., an anti-LDL antibody, anti-denatured LDL antibody, or anti-ApoB antibody, or an antigen-binding fragment thereof, in the case of denatured LDL), or a molecule that specifically binds to the competing molecule (e.g., G-BSA or G-OVA in the case of stimulatory AGEs) (e.g., an anti-BSA antibody or anti-OVA antibody, or an antigen-binding fragment thereof, in the case of stimulatory AGEs), and these can be used as control binding agents.

[0030] As used herein, the term "membrane" refers to a porous or nonporous solid phase that is insoluble in water, preferably at least partially composed of a material capable of binding or retaining biomolecules. The membrane used herein preferably allows sample development by capillary action and may at least partially comprise a material that achieves this. Non-exhaustive examples of materials that may constitute membranes include cellulose, polysaccharides such as SEPHADEX™, glass, polyacryloylmorpholide, silica, controlled pore glass (CPG), polystyrene, polystyrene / latex, polyethylenes such as ultra-high molecular weight polyethylene (UPE), polyamide, polyvinylidine fluoride (PVDF), polytetrafluoroethylene (PTFE; Teflon®), carboxyl-modified Teflon®, nylon, nitrocellulose, and metals and alloys such as gold, platinum, and palladium. Membranes are typically charged and bind to organic materials such as proteins. Membranes significantly improve various analytical processes by making them quantitative.

[0031] As used herein, "modified LDL" is also referred to as "modified LDL" or "modified LDL" (these terms are used interchangeably), and refers to any LDL modification that has various molecular modifications that occur when LDL comes into contact with reactive oxygen species, oxidative enzymes, Fe3+, etc. in the body, or through cell-dependent chemical changes by vascular endothelial cells, macrophages, etc. Representative examples of LDL modifications present in the body include, but are not limited to, oxidized LDL (referred to herein as OxLDL, and examples thereof include fully oxidized LDL (also referred to herein as fu OxLDL) and partially oxidized LDL (also referred to herein as mo OxLDL)), aldehyde-modified LDL such as malondialdehyde-modified LDL (MDA-LDL) and crotonaldehyde (CRA)-modified LDL, acrolein-modified LDL, nonenal-modified LDL, 4-hydroxynonenal (HNE)-modified LDL, hexanoyl (HEL)-modified LDL, small particle LDL (LDL with a diameter of 255 nm or less), glycated LDL, and acetylated LDL (AcLDL). Abnormal oxidized LDL levels are predictive of diseases such as, but not limited to, arteriosclerosis, ischemic heart disease (myocardial infarction, angina pectoris, etc.), cerebrovascular disorders (cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attack, etc.), aortic aneurysm, renal infarction, and hyperlipidemia (see "Today's Clinical Tests 2007-2008," published by Nankodo Co., Ltd.). Commonly used test methods use MDA-LDL (normal range: 10-80 μL) and oxidized phosphatidylcholine (normal range: 8.4 U / mL-17.6 U / mL) as reference substances.

[0032] As used herein, the term "CTLD molecule" is understood to include CTLD-like polypeptides as well as any complexes thereof. Therefore, CTLD molecules are understood to include full-length LOX-1, the full-length LOX-1 extracellular domain (S61-Q273), CTLD14(129-143), CTLD(143-273), etc.As used herein, the terms "CTLD14" and "PR-CTLD14" refer to: (1) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; (2) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, which contains one or several amino acid substitutions, additions, and / or deletions; (3) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, which contains one or several amino acid substitutions, additions, and / or deletions at amino acid positions other than positions 104 and 121, and which exhibits the activity of native LOX-1; (4) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (5) a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (6) a polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule set forth in SEQ ID NO: 1; (7) a polypeptide that hybridizes under stringent conditions with a nucleic acid sequence complementary to the nucleic acid sequence set forth in SEQ ID NO: 1. (8) a polypeptide comprising 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, wherein the amino acids at positions 104 and 121 in the encoded amino acid sequence retain the corresponding amino acids in SEQ ID NO: 2, and the polypeptide exhibits the activity of native LOX-1; (9) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having one or more substitutions, additions, and / or deletions in the nucleic acid sequence shown in SEQ ID NO: 1, and exhibits the activity of native LOX-1; (10) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1; or (11) a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. The above identity or homology is calculated using the sequence analysis tool BLAST (NCBI's BLAST 2.9 (issued March 11, 2019)) with default parameters.Stringent conditions will vary depending on the sequence, and the determination of such conditions is within the skill of one in the art.

[0033] As used herein, the term "advanced glycation end products" (AGEs), also known as AGEs, is a collective term for a diverse range of structures that are the products of protein glycation. While AGEs are produced during food processing and are important for improving taste, they are also produced in vivo, and some of them can cause dysfunction and trigger age-related diseases. They are also known to be involved in the onset and progression of diabetic vascular disease, a vascular complication that significantly impairs the quality of life of diabetic patients. Eye, nerve, and kidney damage caused by vascular complications are known as diabetic retinopathy, neuropathy, and nephropathy (collectively known as the three major complications), respectively, and are characteristic pathologies of diabetic patients. Reducing sugars, such as glucose, react nonenzymatically with amino groups in proteins and amino acids to form glycation products such as Schiff bases or Amadori rearrangement compounds. The reactions up to this stage are reversible and are referred to as the early reactions. Subsequently, advanced glycation end products (ADPs) are formed through complex and irreversible reactions such as condensation, cleavage, and crosslinking. This series of reactions is called glycation. AGEs are also a collective term for structures generated through this process. AGE structures present in living organisms include, but are not limited to, carboxymethyllysine (CML), carboxyethyllysine (CEL), pentosidine, pyrraline, imidazoline, methylglyoxal, and crosslin. The glycation products of albumin, immunoglobulins, and ovalbumin present in plasma are also AGEs and are commonly used in experimental systems. Furthermore, in in vitro experimental systems, glycated versions of BSA (bovine serum albumin), such as R-AGE (BSA glycated with ribose), F-AGE (BSA glycated with fructose), and G-AGE (BSA glycated with glucose), are also commonly used. Hemoglobin A1c, which is used as an indicator of blood sugar control, is an Amadori transition compound and is included in AGEs. Any protein can also be converted into AGEs. For example, CML albumin and CEL albumin, which are included in AGEs, are both AGEs formed by glycation of albumin.Such AGE production reactions can occur in vivo in the circulating blood, the extracellular matrix, and intracellularly. For example, AGEs present in the blood vessels of diabetic patients can be broadly divided into two types: fluorescent and cross-linked (e.g., pentosidine and cross-lin) and non-fluorescent and non-cross-linked (e.g., carboxymethyllysine, pyrraline, and methylglyoxal (MG)-imidazolone). Abnormal AGE levels suggest microangiopathy (nephropathy, retinopathy, neuropathy, etc.) and macroangiopathy (ischemic heart disease, cerebrovascular disease, and arteriosclerosis obliterans). Commonly used test methods use pyrraline (normal range: less than 23 pmol / mL in plasma) and pentosidine (normal range: 0.00915–0.0431 μg / mL in plasma (measured by ELISA)) as reference substances (see "Today's Clinical Tests 2007–2008," published by Nankodo Co., Ltd.).

[0034] In this specification, "irritating advanced glycation end products" or "irritating AGEs" refers to AGEs that are highly associated with disease and have the property of binding strongly to sRAGE. Previously, it was thought that glycation by blood glucose was the primary mechanism of AGE formation. However, it has recently been suggested that glucose-induced glycation takes a long time and that glucose-glycated AGEs are less irritating to the body. Excess glucose is metabolized in the polyol metabolic pathway to produce glyceraldehyde (Glycer), and also produces glyoxal (GO) and glycolaldehyde (Glycol) through oxidation. These are highly reactive and rapidly generate AGEs, and their glycation products have been reported to be highly biotoxic. It has been suggested that liver diseases (e.g., NASH) are particularly closely associated with proteins modified by glyceraldehyde (Glycer-AGEs).

[0035] As used herein, the term "AGE molecule" refers to any molecule included in the above-mentioned AGEs. Examples of AGEs include, but are not limited to, Lys-AGE (glutaraldehyde-modified lysine-modified AGE), glucose-modified AGE (G-AGE), ribose-modified AGE (R-AGE), fructose-modified AGE (F-AGE), or variants or complexes thereof.

[0036] As used herein, the term "molecule exhibiting AGE-like activity" refers to a molecule that has at least one of the activities of the above-mentioned AGEs (referred to as "AGE-like activity" in this specification). Such AGE-like activity includes, but is not limited to, binding activity (ligand activity) to RAGE.

[0037] As used herein, the term "AGE receptor" is also referred to as RAGE and includes: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4; (2) a polypeptide comprising an amino acid sequence containing one or more amino acid substitutions, additions, and / or deletions in the amino acid sequence shown in SEQ ID NO: 4, and exhibiting the activity of native RAGE; (3) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and exhibiting the activity of native RAGE; (4) a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and exhibiting the activity of native RAGE; (5) a polypeptide comprising the amino acid sequence encoded by the nucleic acid molecule shown in SEQ ID NO: 3; (6) a nucleic acid sequence complementary to the nucleic acid sequence shown in SEQ ID NO: 3. (7) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent conditions with the nucleic acid sequence set forth in SEQ ID NO: 3 and exhibits the activity of native RAGE; (8) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 90% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3 and exhibits the activity of native RAGE; and (9) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having at least 80% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3 and exhibits the activity of native RAGE. The identity or homology is calculated using the sequence analysis tool BLAST (NCBI's BLAST 2.9 (published March 11, 2019)) with default parameters. Stringent conditions vary depending on the sequence, and determining such conditions is within the skill of one of ordinary skill in the art. RAGE was also identified from bovine lung in 1992 and is a type I membrane protein with a molecular weight of approximately 35 kDa (the complete RAGE after glycosylation has a molecular weight of 55 kDa) that belongs to the immunoglobulin superfamily and binds to AGEs.The extracellular domain of RAGEs is composed of three immunoglobulin-fold domains: one V-type immunoglobulin domain followed by two C-type immunoglobulin domains (C1 and C2 regions). RAGE also contains a single 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 essential for ligand binding, and the cytoplasmic domain is essential for RAGE-mediated intracellular signaling. Because RAGE also has disulfide bonds within each domain, the mutant RAGE-surfactant complexes of the present disclosure preferably retain the cysteine ​​residues corresponding to positions 38, 99, 144, 208, 259, and 301 in the amino acid sequence of SEQ ID NO: 6. RAGE is expressed at low levels in normal tissues and the vasculature. However, this receptor is upregulated at sites where its ligand accumulates. RAGE expression is increased in endothelial cells, smooth muscle cells, pericytes, renal mesangial cells, and infiltrating mononuclear phagocytes in the diabetic vasculature. RAGE expression is also increased at pathological sites, such as atherosclerotic plaques, where AGEs accumulate. AGE-RAGE interactions alter cellular properties important 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 the expression of cytokines and growth factors and induces cell migration in response to soluble AGEs, whereas haptotaxis occurs with immobilized ligands.

[0038] As used herein, the terms "RAGE ligand recognition domain" and "sRAGE (soluble receptor for Advanced Glycation End products)" are used interchangeably and refer to the domain recognized by a RAGE ligand. Specifically, sRAGE, i.e., the RAGE ligand recognition domain, refers to all or a portion of the extracellular domain of RAGE. sRAGE typically consists of positions 22 to 332 of SEQ ID NO: 6 or SEQ ID NO: 4, but is not limited thereto.

[0039] As used herein, the term "RAGE-like polypeptide" includes polypeptides designated as "RAGE8," "mRAGE8," "RAGE1," "mRAGE1," "RAGE2," "mRAGE2," "RAGE3," "mRAGE3," "RAGE4," "mRAGE4," "RAGE7," "mRAGE7," "RAGE143," "mRAGE143," "RAGE223," "mRAGE223," "RAGE226," and "mRAGE226," or variants thereof. These descriptions are disclosed in, for example, JP 2013-209330, the contents of which are incorporated herein by reference as appropriate.

[0040] As used herein, the term "RAGE molecule" is understood to include RAGE-like polypeptides as well as any complexes thereof. Therefore, RAGE molecules are understood to encompass RAGE-like polypeptides, such as RAGE (full-length), the RAGE extracellular domain (positions 22-332 of SEQ ID NO: 4), RAGE143, RAGE223, and RAGE226. RAGE molecules also encompass RAGE (mini-RAGE) lacking an entire or partial domain among the three domains constituting RAGE. Mini-RAGE also encompasses mini-RAGE, a RAGE-like polypeptide.

[0041] As used herein, molecules comprising a "RAGE ligand recognition region" include "RAGE molecules" other than full-length RAGE (including "RAGE-like polypeptides"), such as "RAGE8," "mRAGE8," "RAGE1," "mRAGE1," "RAGE2," "mRAGE2," "RAGE3," "mRAGE3," "RAGE4," "mRAGE4," "RAGE7," "mRAGE7," "RAGE143," "mRAGE143," "RAGE223," "mRAGE223," "RAGE226," and "mRAGE226," as well as the RAGE extracellular region (positions 22-332 of SEQ ID NO: 4), etc.

[0042] The RAGE-like polypeptide may contain unnatural amino acids, amino acid analogs, amino acid derivatives, etc., as long as it retains the activity of native RAGE.

[0043] In the above-mentioned RAGE-like polypeptides, since the formation of intramolecular disulfide bonds is important, it is preferable that the cysteines corresponding to positions 38, 99, 144, 208, 259 and 301 of the amino acid sequence of SEQ ID NO: 4 are retained.

[0044] As used herein, a "ligand" is a binding partner for a specific receptor or family of receptors. A ligand may be an endogenous ligand for a receptor, or alternatively, a synthetic ligand for a receptor, such as a drug, drug candidate, or pharmacological tool.

[0045] As used herein, the term "antibody" broadly includes polyclonal, monoclonal, multispecific, chimeric, and anti-idiotypic antibodies, as well as functional fragments thereof (e.g., F(ab')2 and Fab fragments), and other recombinantly produced conjugates or functional equivalents (e.g., chimeric, humanized, multifunctional, bispecific, or oligospecific antibodies, single-chain antibodies (scFv), diabodies, single-chain (Fv)2, and scFv-Fc). Furthermore, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, and alpha-galactosidase. Furthermore, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, and alpha-galactosidase. When used in the narrow sense, the term "antibody" refers to a full-length antibody (e.g., a polyclonal antibody, a monoclonal antibody, etc.), and may also be referred to as a variant or an antigen-binding fragment. The antibody used in the present disclosure may be of any origin, type, shape, etc., as long as it binds to its target. Specifically, it can be produced based on known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies. In the present disclosure, a single-chain antibody is used. The antibody preferably binds to its target in a discriminatory or specific manner. A variant antibody may be bound to various molecules, such as polyethylene glycol. A variant antibody can be obtained by chemically modifying the antibody using known techniques.

[0046] As used herein, the term "single chain antibody" is also referred to as "scFv (single chain Fv)" and refers to a single chain antibody consisting of the variable regions of the heavy and light chains (V H and V L) linked with an appropriate linker peptide. Such a construct can be constructed at the genetic level and introduced into E. coli using a protein expression vector to express the single-chain antibody protein.

[0047] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The length of a fragment can be varied appropriately depending on its purpose. For example, the lower limit of the length for a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically recited herein (e.g., 11) may also be suitable as the lower limit. Furthermore, for a polynucleotide, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically recited herein (e.g., 11) may also be suitable as the lower limit. As used herein, the length of a polypeptide or polynucleotide can be expressed in terms of the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the above numbers as upper or lower limits are intended to include several positions above or below that number (or, for example, 10% above or below) as long as they have the same function. The length of a fragment useful herein can be determined by whether at least one function of the full-length protein that serves as the basis for the fragment is retained.

[0048] As used herein, the term "homology" of genes refers to the degree of identity between two or more gene sequences. Thus, the higher the homology between two genes, the greater the identity or similarity between their sequences. Whether two genes are homologous can be determined by direct comparison of the sequences or, in the case of nucleic acids, by hybridization under stringent conditions. When two gene sequences are directly compared, the genes are homologous if the DNA sequences between the gene 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% identical.

[0049] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0050] In this specification, comparison of similarity, identity, and homology of amino acid sequences and nucleotide sequences is calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.9 (published March 11, 2019). The identity value in this specification usually refers to the value obtained when aligned under default conditions using the above-mentioned BLAST. However, if a higher value is obtained by changing the parameters, the highest value is used as the identity value. If identity is evaluated in multiple regions, the highest value among them is used as the identity value.

[0051] As used herein, the term "variant" refers to a substance, such as an original polypeptide or polynucleotide, that has been partially altered. Examples of such variants include substitution variants, addition variants, deletion variants, truncated variants, and allelic variants. An allele refers to a genetic variant that resides at the same locus and is distinct from one another. Thus, an "allelic variant" refers to a variant that is allelic to a given gene. A "species homolog" refers to a gene within a species that shares homology with a given gene at the amino acid or nucleotide level (preferably 60% or more homology, more preferably 80% or more, 85% or more, 90% or more, or 95% or more homology). Methods for obtaining such species homologs are clear from the description herein. An "ortholog," also known as an orthologous gene, refers to a gene derived from speciation of two genes from a common ancestor. For example, in the hemoglobin gene family, which has a multigene structure, the human and mouse alpha hemoglobin genes are orthologs, while the human alpha hemoglobin and beta hemoglobin genes are paralogs (genes resulting from gene duplication). Because orthologs are useful for estimating molecular phylogenetic trees, orthologs may also be useful in the present disclosure.

[0052] As used herein, the term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Methods for modifying such sequences include cleavage with restriction enzymes, ligation with DNA polymerase, Klenow fragment, DNA ligase, and site-specific base substitution using synthetic oligonucleotides (site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)). Modifications can also be made by other methods commonly used in molecular biology. Due to the degeneracy of the genetic code, a large number of 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 an alanine is specified by a codon, that codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such variations of nucleic acids are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of that nucleic acid. It is understood in the art that 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) can be altered to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such alterations are made to avoid substitutions of cysteine, an amino acid that significantly affects the conformation of a polypeptide.

[0053] Certain amino acids can be substituted for other amino acids in protein structures, such as the binding site of a ligand molecule, without any significant reduction or loss of interactive binding ability. The biological function of a protein is determined by the protein's interactive ability and properties. Therefore, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in a protein that still maintains its original properties after the substitution. Therefore, various modifications can be made in the peptides disclosed herein or the corresponding DNA encoding the peptides without any significant loss of biological usefulness.

[0054] Such nucleic acids can be obtained by the well-known PCR method or can be chemically synthesized, which may be combined with, for example, site-directed mutagenesis or hybridization.

[0055] When designing such modifications, the hydrophobicity index of amino acids can be taken into consideration. The importance of the hydrophobic amino acid index in providing interactive biological function to proteins is generally recognized in the art (Kyte, J and Doolittle, RFJ Mol. Biol. 157(1):105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the resulting protein, and then determines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on its 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); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0056] It is well known in the art that an amino acid can be substituted with another amino acid having a similar hydrophobicity index and still produce a protein having a similar biological function (e.g., a protein equivalent in ligand binding ability). 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 amino acid substitutions based on hydrophobicity are efficient. As described in U.S. Patent No. 4,554,101, the following hydrophilicity indexes are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and still provides a biological equivalent. In such amino acid substitutions, the hydrophilicity index is preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0057] In the present disclosure, the term "conservative substitution" refers to an amino acid substitution in which the hydrophilicity index or / and hydrophobicity index of the original amino acid and the substituted amino acid are similar as described above. Examples of conservative substitutions are well known to those skilled in the art, and include, but are not limited to, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0058] As used herein, in addition to amino acid substitution, amino acid addition, deletion, or modification can also be performed to prepare functionally equivalent polypeptides. Amino acid substitution refers to substituting one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3, amino acids into the original peptide. Amino acid addition refers to adding one or more, 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 deleting 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 (e.g., acetylation), and the like. The substituted or added amino acids may be natural amino acids, unnatural amino acids, or amino acid analogs. Natural amino acids are preferred.

[0059] As used herein, "substitution, addition, and / or deletion" of a polypeptide or polynucleotide refers to the replacement, addition, or removal of an amino acid or its substitute, or a nucleotide or its substitute, from the original polypeptide or polynucleotide, respectively. Techniques for such substitution, addition, and / or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis. These changes in the reference nucleic acid molecule or polypeptide can occur at the 5' or 3' end of the nucleic acid molecule, or at the amino or carboxy terminal end of the amino acid sequence representing the polypeptide, or anywhere between these terminal ends, and can be individually dispersed among residues in the reference sequence, so long as the desired function (e.g., RAGE recognition ability) is maintained. The number of substitutions, additions, or deletions may be one or more, and can be as large as necessary, as long as the desired function is maintained in the variant having the substitution, addition, or deletion. For example, such number may be one or several, and preferably within 20%, 15%, 10%, or 5% of the overall length, or 150 or less, 100 or less, 50 or less, 25 or less, etc.

[0060] As used herein, the term "tag sequence" refers to a substance for selecting molecules through a specific recognition mechanism such as receptor-ligand, more specifically, a substance that acts as a binding partner for binding a specific substance (e.g., having a relationship such as biotin-avidin or biotin-streptavidin). Thus, for example, a specific substance bound to a tag sequence can be selected by contacting the substance with a substrate to which a binding partner of the tag sequence is bound. Such tag sequences are well known in the art. Representative tag sequences include, but are not limited to, myc tags, His tags, HA tags, Avi tags, etc.

[0061] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term may also encompass multiple polypeptide chains assembled into complexes. The term also encompasses naturally occurring or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling component). The definition also encompasses, for example, polypeptides containing one or more analogs of an amino acid (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art.

[0062] As used herein, "amino acids" may be natural or non-natural, as long as they satisfy the objectives of this disclosure. As used herein, "amino acid derivatives" or "amino acid analogs" refer to amino acids that differ from naturally occurring amino acids but have the same function as the original amino acid. Such amino acid derivatives and analogs are well known in the art. As used herein, it is understood that amino acid derivatives and analogs can be used as substitutes for amino acids as long as they can provide the same biological function. As used herein, "natural amino acids" refer to the L-isomers of natural amino acids. Natural amino acids include glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine, and lysine. Unless otherwise specified, all amino acids referred to herein are in the L-form, although forms using D-form amino acids are also within the scope of this disclosure. As used herein, the term "unnatural amino acid" refers to an amino acid 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-forms of homoarginine, and D-phenylalanine. As used herein, the term "amino acid analog" refers to a molecule that is not an amino acid but mimics the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, and 2-methylglutamine. An amino acid mimetic refers to a compound that has a structure different from the general chemical structure of an amino acid but functions in a manner similar to a naturally occurring amino acid.

[0063] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0064] As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. This term also includes "oligonucleotide derivatives" or "polynucleotide derivatives." "Oligonucleotide derivatives" or "polynucleotide derivatives" refer to oligonucleotides or polynucleotides that contain derivatives of nucleotides or have unusual internucleotide bonds, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond in the oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which the uracil in the oligonucleotide has been substituted with C-5 propynyl uracil, oligonucleotide derivatives in which the uracil in the oligonucleotide has been substituted with C-5 thiazole uracil, oligonucleotide derivatives in which the cytosine in the oligonucleotide has been substituted with C-5 propynyl cytosine, oligonucleotide derivatives in which the cytosine in the oligonucleotide has been substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in the DNA has been substituted with 2'-O-propyl ribose, and oligonucleotide derivatives in which the ribose in the oligonucleotide has been substituted with 2'-methoxyethoxy ribose. Unless otherwise indicated, a particular nucleic acid sequence is also intended to encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (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)).

[0065] As used herein, a "nucleotide" may be natural or non-natural. A "nucleotide derivative" or a "nucleotide analog" refers to a nucleotide that is different from a 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 not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0066] As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. A particular nucleic acid sequence also encompasses "splice variants." Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any proteins encoded by splice variants of that nucleic acid. As the name suggests, "splice variants" are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced ​​such that different (alternate) nucleic acid splice products encode different polypeptides. The mechanism for producing splice variants varies but includes alternative splicing of exons. Alternative polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of splice products, are included in this definition.

[0067] As used herein, "gene" refers to a factor that determines a genetic trait. It is usually arranged in a specific order on a chromosome. A gene that determines the primary structure of a protein is called a structural gene, and a gene that controls its expression is called a regulatory gene. As used herein, "gene" can refer to "polynucleotide," "oligonucleotide," and "nucleic acid," and / or "protein," "polypeptide," "oligopeptide," and "peptide."

[0068] As used herein, "phosphate buffered saline (PBS)" refers to an aqueous solution containing NaCl, KCl, NaHPO, and KHPO, with a pH of 7 to 8. The concentration and pH of each component can be adjusted appropriately depending on the intended use. As used herein, "PBS(+)" refers to the inclusion of calcium ions and magnesium ions, while "PBS(-)" refers to the absence of calcium ions and magnesium ions. However, unless otherwise specified, "PBS" refers to "PBS(-)." Dulbecco's PBS(-) can be used as a representative solution. Dulbecco's PBS(-) has the following composition: NaCl 8 g, KCl 0.2 g, NaHPO 1.15 g, KHPO 0.2 g / L (pH 7.4).

[0069] As used herein, the term "receptor" refers to a biological structure with one or more binding domains that reversibly and specifically complex with one or more ligands, where the complexation comprises a biological structure. Receptors can be located entirely outside the cell (extracellular receptors), within the cell membrane (but with portions of the receptor facing the extracellular environment and cytosol), or entirely within the cell (intracellular receptors). They can also function independently of the cell. Receptors in the cell membrane allow the cell to communicate with spaces outside its boundaries (e.g., signal transduction) and function in the transport of molecules and ions into and out of the cell. As used herein, receptors can be full-length receptors or receptor fragments.

[0070] As used herein, the term "antigen-antibody reaction" is used in the broadest sense used in the art, and particularly refers to a reaction based on the specific binding between an antigen and an antibody. We also provide reagents and methods for detecting and quantifying antigens in a sample using an immunoblot (Western blot) format as a detection system.

[0071] sample As used herein, "silkworm" refers to the ordinary meaning of silkworm, a species of insect belonging to the order Lepidoptera and the family Bombycidae. Its official Japanese name is silkworm moth (scientific name: Bombyx mori), and while "silkworm" refers to the larvae, it generally refers to the entire species. Silkworms feed on mulberry trees, producing silk and creating pupal cocoons. Silkworms, also known as domesticated silkworms, are not wild-caught insects. Silkworms are believed to have descended from the mulberry silkworm (Bombyx mandarina), which lives in East Asia. While silkworms and mulberry silkworms are considered scientifically distinct species, hybrids between them are reproductively capable. In this specification, "silkworms" includes mulberry silkworms. As used herein, "organisms capable of adding sugar chains similar to those of silkworms" refers to organisms capable of adding sugar chains similar to those of silkworms, and may include transgenic organisms containing genes encoding enzymes that add sugar chains similar to those of silkworms.

[0072] As used herein, the term "silk gland" refers to a pair of organs present in the body of mature silkworms, one on the left and one on the right, which convert a large amount of protein (amino acids) ingested from mulberry leaves into two types of silk proteins (fibroin and sericin). The pair of silk glands secrete liquid silk, which serves as the raw material for cocoon threads. The silk gland is divided into three parts: the posterior silk gland, the middle silk gland, and the anterior silk gland. In the present disclosure, any of the silk glands can be used for synthesis, but in consideration of handling after synthesis, the posterior silk gland and the middle silk gland are usually used, and preferably the middle silk gland, but this is not limiting. Furthermore, silk can be expressed throughout the body and then collected from the whole body, or it can be collected from the cocoon after cocoon formation.

[0073] The posterior silk gland is a long, thin part at the very back of the silkworm that can stretch to about 20 cm. This gland synthesizes the fibroin protein that later becomes the core of the cocoon thread.

[0074] The middle silk gland is a thick, S-shaped section in the center that can stretch out to about 6 cm. It concentrates and stores the fibroin protein sent from the posterior silk gland, shaping it into a form that is easy to turn into fiber. It also secretes another silk protein, sericin, which acts as an adhesive to hold the fibroin protein together when the cocoon thread is spun out.

[0075] The anterior silk gland is a thin tube connected to the spinneret, approximately 4 cm long, that becomes thinner as it approaches the tip. The liquid fibroin protein molecules are stretched and aligned in a certain direction, and as they gather together, further moisture is removed. At the tip of the tube, they merge with another pair of tubes to form a single thread, which is then expelled from the spinneret to become a single cocoon thread.

[0076] Silkworms stop eating mulberry leaves towards the end of their fifth instar (mature silkworm). The body of a mature silkworm is filled with a pair of organs (silk glands) that store a syrup-like liquid (liquid silk), which is the raw material for cocoon thread. The silk glands are connected to the spinnerets at the mouth of the silkworm via thin spinnerets. As the liquid silk passes through the thin spinnerets, it is stretched and hardens, becoming cocoon thread. Furthermore, the larva sticks the thread it spits out from the spinnerets to nearby objects, moves its head and thorax in a figure-eight shape, and pulls it, a series of movements that allows cocoon thread to be drawn out one after another from the silk gland.

[0077] As used herein, "silkworm-type sugar chain" refers to a sugar chain structure specific to glycoproteins produced by silkworms, and typically includes a trimannosyl core (itself), an oligomannose-type sugar chain, a complex-type sugar chain, or a hybrid type thereof. In the present disclosure, since silkworm-type glycoproteins are produced using the middle silk gland, unless otherwise specified, "silkworm-type sugar chain" refers to the specific sugar chain type produced in this middle silk gland. For example, such silkworm-type sugar chains have a core structure formed by the binding of two N-acetylglucosamine (GlcNAc) molecules linked to asparagine (Asn), followed by the binding of three mannose (Man) molecules (referred to as a trimannosyl core, shown in formula (1) below), from which branched structures are formed, to which various sugar chains are further bound.

[0078] As used herein, a "corresponding" amino acid or nucleic acid refers to an amino acid or nucleotide in a polypeptide or polynucleotide molecule that has or is predicted to have the same function as a given amino acid or nucleotide in a reference polypeptide or polynucleotide. In particular, in the case of an enzyme molecule, this refers to an amino acid that is located at a similar position in the active site and contributes similarly to catalytic activity. For example, in the case of an antisense molecule, this may be a similar portion in an orthologue corresponding to a specific portion of the antisense molecule. The corresponding amino acid may be, for example, a specific amino acid that is cysteinylated, glutathionylated, forms an S-type disulfide bond, oxidized (e.g., oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, or the like. Alternatively, the corresponding amino acid may be an amino acid responsible for dimerization. Such a "corresponding" amino acid or nucleic acid may be a region or domain spanning a certain range. Therefore, in such cases, it is referred to herein as a "corresponding" region or domain.

[0079] As used herein, a "corresponding" gene (e.g., a polypeptide molecule or polynucleotide molecule) refers to a gene (e.g., a polypeptide molecule or polynucleotide molecule) that has or is predicted to have the same function in a given species as a given gene in a reference species. When multiple genes with such function exist, the term refers to genes that have the same evolutionary origin. Thus, a gene corresponding to a given gene may be its ortholog. Thus, for mouse and rat RAGE (or the soluble form of sRAGE), a corresponding RAGE (sRAGE or the soluble form of sRAGE) can be found in humans, respectively. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in a given animal (e.g., a mouse), or a reference gene (e.g., RAGE or the soluble form of sRAGE) for the corresponding gene, can be found by searching a sequence database for that animal (e.g., a human or rat) using the sequence of that animal as a query sequence.

[0080] As used herein, the term "biological function," when referring to a gene or its associated nucleic acid molecule or polypeptide, refers to a specific function that the gene, nucleic acid molecule, or polypeptide may have in a living organism, including, but not limited to, the production of specific antibodies, enzymatic activity, and the conferring of resistance. In the present disclosure, the term "biological function" refers to, but is not limited to, the function of RAGE recognizing markers such as hemopexin. As used herein, biological function can be exerted through "biological activity." As used herein, "biological activity" refers to the activity that a certain factor (e.g., polynucleotide, protein, etc.) may have in a living organism, including the ability to exert various functions (e.g., transcription-promoting activity), including the activation or inactivation of another molecule through interaction with another molecule. When two factors interact, their biological activity is determined by the binding between the two molecules and the resulting biological change. For example, if one molecule is co-precipitated when the other molecule is co-precipitated with an antibody, the two molecules are considered to be bound. Therefore, observing such co-precipitation is one method of assessment. For example, if a factor is an enzyme, its biological activity includes its enzymatic activity. In another example, if a factor is a ligand, it includes the binding of the ligand to a corresponding receptor. Such biological activities can be measured by techniques well known in the art.

[0081] Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either direct or indirect); or affect a response (i.e., have a measurable effect in response to some exposure or stimulus), including, for example, the affinity of a compound to bind directly to a polypeptide or polynucleotide of the present disclosure, or, for example, a measure of the amount of an upstream or downstream protein or other similar function after some stimulus or event.

[0082] As used herein, the term "subject" refers to a living organism (e.g., a human) that is the subject of diagnosis or detection, etc., in the present disclosure.

[0083] As used herein, the term "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.).

[0084] As used herein, the terms "drug," "agent," and "factor" (all of which correspond to the English term "agent") are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, or electricity) that can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands), etc.), and composite molecules thereof. Typical examples of factors specific to a polynucleotide include, but are not limited to, polynucleotides that are complementary to the sequence of the polynucleotide with a certain degree of sequence homology (e.g., 70% or more sequence identity), and polypeptides such as transcription factors that bind to promoter regions. Typical examples of factors specific to a polypeptide include, but are not limited to, antibodies or derivatives or analogs thereof (e.g., single-chain antibodies) specifically directed against the polypeptide, specific ligands or receptors when the polypeptide is a receptor or ligand, and substrates when the polypeptide is an enzyme.

[0085] As used herein, the term "interaction," when referring to two substances, refers to the mutual exertion of forces (e.g., intermolecular forces (van der Waals forces), hydrogen bonds, hydrophobic interactions, etc.) between one substance and the other. Typically, two substances that have interacted are in an associated or bonded state.

[0086] As used herein, the term "binding" refers to a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or combinations thereof. Binding includes ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. A physical interaction (binding) can be direct or indirect, where indirect binding is mediated through or caused by the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or caused by the effect of another protein or compound and does not involve any other substantial chemical intermediate.

[0087] As used herein, "contacting" means bringing a compound into physical proximity, either directly or indirectly, to a polypeptide or polynucleotide that can function as a marker, ligand, etc. of the present disclosure. The polypeptide or polynucleotide can be present in any number of buffers, salts, solutions, etc. Contacting can include placing the compound in, for example, a beaker, microtiter plate, cell culture flask, or microarray (e.g., gene chip), etc., that contains a polypeptide encoding a nucleic acid molecule or fragment thereof.

[0088] In one aspect, the present disclosure is used to detect oxidized LDL or irritating AGEs, which are useful for diagnosing diseases (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's disease) and evaluating the effectiveness of treatments.

[0089] As used herein, "liver disease" refers to any disease of the liver. The liver disease targeted by the present invention can be any liver disease, but liver diseases can be chronic fatty liver disease or acute fatty liver disease, which can be inflammatory diseases, lifestyle-related inflammatory diseases, non-alcoholic diseases, or non-viral diseases. The present invention can be useful for diagnosing diseases such as non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), as well as diseases that are more advanced stages of NASH, such as cirrhosis and hepatocellular carcinoma.

[0090] 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 progression are long-term. Factors that cause chronic disease include, but are not limited to, age, gender, lifestyle, genetic factors, obesity, various hormonal abnormalities, and the intake of certain drugs. Therefore, chronic fatty liver disease is a completely different disease from "acute" fatty liver disease, and has different pathologies, causes, and treatments and prevention methods.

[0091] As used herein, term " inflammatory disease " refers to the disease that accompanies inflammation, and when fatty liver disease is referred to as inflammatory disease, it refers to any fatty liver disease that accompanies inflammation.It is understood that fatty liver disease that is inflammatory disease also includes non-alcoholic steatohepatitis (NASH) and its corresponding alcoholic steatohepatitis.

[0092] As used herein, the term "lifestyle-related inflammatory disease" refers to a disease associated with inflammation and whose primary cause is lifestyle. Lifestyle in the present invention includes, but is not limited to, exercise amount, nutritional balance, smoking, alcohol intake, and sleep duration. The term "inflammatory disease" is used in the sense commonly used in the art, and refers to a disease characterized by a local tissue reaction to injury in the body. In a specific embodiment, the inflammatory disease in the present invention is a liver-related inflammatory disease.

[0093] As used herein, the term "non-alcoholic disease" refers to a general term for diseases that are not primarily caused by alcohol consumption. Subjects suffering from non-alcoholic diseases include not only subjects who do not consume alcohol at all, but also subjects who consume small amounts of alcohol (men consuming less than 30g of pure ethanol per day, and women consuming less than 20g of pure ethanol per day). Non-alcoholic diseases are also collectively referred to as non-alcoholic fatty liver disease (NAFLD), and representative non-alcoholic diseases include non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis (NASH).

[0094] As used herein, the term "nonalcoholic fatty liver disease (NAFL)" is used in the sense commonly used in the art to refer to a nonalcoholic disease characterized by the accumulation of fat in the liver, but without the infiltration of inflammatory cells into the liver. Among fatty liver diseases, NAFL has relatively mild symptoms and a good prognosis, but it can progress to more severe diseases such as NASH or cirrhosis. In contrast to nonalcoholic fatty liver disease, there is also a disease called "alcoholic fatty liver," which is primarily caused by alcohol consumption. While nonalcoholic fatty liver and alcoholic fatty liver differ in the presence or absence of alcohol consumption, the pathologies of these fatty liver diseases are similar, and it has been reported that these differences disappear 20 years after the onset of the disease.

[0095] As used herein, the term "nonalcoholic steatohepatitis (NASH)" is used in the sense commonly used in the art to refer to a liver disease accompanied by fat accumulation in hepatocytes, hepatocyte ballooning, increased apoptosis, infiltration of inflammatory cells into the central vein area of ​​the liver, a phenomenon in which fatty liver cells are surrounded, phagocytosed, and processed by macrophages (hCLS), and deposition of excessive extracellular matrix in the liver.

[0096] As used herein, the term "Matteoni 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 state of fatty liver disease. The Matteoni classification is a method for classifying NAFLD into four types, Type 1 to Type 4, based on the presence or absence of (1) hepatocyte steatosis, (2) inflammatory cell infiltration, (3) hepatocyte ballooning, (4) liver fibrosis, and (5) Mallory-Denk bodies in liver tissue. Matteoni Type 1 is defined as hepatocyte steatosis only, Type 2 as hepatocyte steatosis with inflammatory cell infiltration only, Type 3 as hepatocyte ballooning, and Type 4 as hepatic fibrosis in addition to Type 3. Types 3 and 4 are diagnosed as NASH.

[0097] As used herein, the term "liver fibrosis" refers to the formation of excessive fibrous connective tissue during liver repair or response processes. While fibrosis itself does not cause symptoms, severe fibrosis can lead to cirrhosis and complications, resulting in the onset of symptoms. There are many types of fibrosis and many causes. For example, in NASH, fibrosis is observed when it progresses to type 4. At this stage, the risk of progression to cirrhosis or liver cancer must also be considered. Furthermore, fibrosis (which can be described as acute fibrosis) can also be caused by chemicals (e.g., carbon tetrachloride), but because fat accumulation in the liver is not observed, its nature differs from the fibrosis in chronic fatty liver disease (which can be described as chronic fibrosis) of the present invention. Without wishing to be bound by theory, acute fibrosis and chronic fibrosis have completely different pathological aspects, and therefore their prevention and treatment are completely different pathologies. Therefore, the findings obtained cannot be used interchangeably in the development of preventive or therapeutic drugs.

[0098] The fibrotic tissue that replaces hepatocytes does not have the function of hepatocytes. Furthermore, the fibrotic tissue may obstruct blood flow to and within the liver, restricting the blood supply to hepatocytes and causing hepatocyte death, which may lead to further progression of fibrosis. The present invention can also predict and diagnose such fibrosis.

[0099] As used herein, "diagnosis" refers to identifying various parameters associated with a disease, disorder, condition, etc. in a subject and determining the current or future state of such a disease, disorder, or condition. Using the methods, devices, and systems disclosed herein, the internal state can be examined, and such information can be used to select various parameters, such as the disease, disorder, condition, and treatment or prevention formulation or method to be administered in the subject. In the narrow sense, "diagnosis" herein refers to diagnosing the current state, but in a broad sense, it also includes "predictive diagnosis," "pre-diagnosis," etc. Early diagnosis is sometimes referred to as "early diagnosis."

[0100] In particular, the terms "predictive diagnosis" and "preliminary diagnosis" are used interchangeably herein. When referring to liver disease, diabetes, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, and other diabetic complications using molecules capable of recognizing LDL or AGEs (e.g., CTLD14, sRAGE), these terms refer to the detection of pre-onset stages of diabetes, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, and other diabetic complications. These terms also include determining the risk of future onset and assessing the risk of developing diabetes for the purpose of preventing liver disease, diabetes, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, and other diabetic complications. By using the methods, kits, compositions, detection agents, diagnostic agents, systems, and the like disclosed herein, the state of the body can be examined in advance, and such information can be used to select various parameters, such as the disease, disorder, condition, and the formulation or method to be administered for treatment or prevention, in a subject. As used herein, "predictive diagnosis" and "preliminary diagnosis" partially overlap with the concept of "early diagnosis," as they also encompass diagnosis at a stage that cannot be diagnosed by other conventional methods.

[0101] The diagnostic method of the present disclosure is, in principle, industrially useful because it can utilize substances excreted from the body and can be carried out without the involvement of medical professionals such as doctors. In this specification, to clarify that the method can be carried out without the involvement of medical professionals such as doctors, it is sometimes referred to as "assisting" "predictive diagnosis, advance diagnosis, or diagnosis." In a broad sense, "diagnosis" also includes evaluating the effectiveness of treatment.

[0102] As used herein, the term "detection agent" broadly refers to any agent capable of detecting a substance of interest (eg, disease-related oxidized LDL or irritating AGEs).

[0103] As used herein, the term "diagnostic agent" broadly refers to any factor that can diagnose a condition of interest (e.g., a disease (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's dementia)).

[0104] As used herein, "measurement" is used in the usual sense in the art and refers to measuring and determining the amount of a certain object. As used herein, "detection" is used in the usual sense in the art and refers to testing and finding a substance, component, etc., "identification" refers to the act of identifying an object within an existing classification system related to that object, and when used in the chemistry field, refers to determining the chemical identity of the target substance (e.g., determining its chemical structure), and "quantification" refers to determining the amount of the target substance present. As used herein, "detection or quantification by conjugate formation with (a molecule)" refers to the detection or quantification of the target object, using as an indicator whether the target object forms a conjugate with another entity. This can be done using conjugate formation as an indicator or inhibition of conjugate formation (using a competing molecule).

[0105] As used herein, the term "treatment" refers to, with respect to a certain disease or disorder, preventing the disease or disorder from worsening when such a condition has developed, preferably maintaining the status quo, more preferably alleviating the condition, and even more preferably causing the disease or disorder to disappear.

[0106] As used herein, "prevention" refers to preventing a certain disease (such as dyslipidemia, diabetic complications, liver disease, and Alzheimer's dementia) or disorder from occurring before that state is reached. By performing the predictive diagnosis or advance diagnosis of the present disclosure, diseases or disorders associated with oxidized LDL or irritating AGEs can be prevented, or preventive measures can be taken. By performing the predictive diagnosis or advance diagnosis of the present disclosure, diabetic complications such as diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy can be prevented, or preventive measures can be taken.

[0107] (Preferred embodiment) Although the following description of preferred embodiments is given, it should be understood that these embodiments are merely examples of the present disclosure and that the scope of the present disclosure is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications, changes, etc. within the scope of the present disclosure by referring to the following preferred examples. Those skilled in the art can combine any of these embodiments as appropriate.

[0108] (Device or Kit) In one aspect, the present disclosure provides a device or kit for detecting or quantifying an abnormal form of a biomarker molecule by forming a conjugate with the biomarker molecule, the device or kit comprising a membrane that develops a sample by capillary action, wherein the membrane comprises a detection section comprising a detection binding agent that specifically binds to the biomarker molecule or its competitor molecule, and a control section comprising a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with the abnormal form of the biomarker molecule or its competitor molecule, and the kit or device comprises a sample contact section and fluorescent nanoparticles as part of the membrane or as separate elements.

[0109] In some embodiments, the membrane may include, from upstream to downstream, a detection portion and a control portion, in that order.

[0110] In some embodiments, the sample contact portion, the detection portion, and the control portion may be arranged or connected to each other so that the sample penetrates into each other by capillary action.

[0111] In some embodiments, the biomarker molecule can be LDL or AGEs, and the abnormal form of the biomarker molecule can be modified LDL or irritating AGEs.

[0112] In some embodiments, the binding molecule may be CTLD14 or sRAGE.

[0113] In some embodiments, when the target to be detected is denatured LDL, the detectable binding agent may be an anti-LDL antibody, an anti-denatured LDL antibody, an anti-ApoB antibody, or an antigen-binding fragment thereof; when the target to be detected is irritating AGEs, the detectable binding agent may be an anti-BSA antibody or an anti-OVA antibody, or an antigen-binding fragment thereof.

[0114] In some embodiments, the kits or devices of the present disclosure may or may not further include a competitor molecule of the biomarker molecule.

[0115] In certain embodiments, the biomarker molecule is LDL, the abnormal form of the biomarker molecule is denatured LDL, the binding molecule is CTLD14, and the detectable binding agent can be an anti-LDL antibody, an anti-denatured LDL antibody, or an anti-ApoB antibody, or an antigen-binding fragment thereof.

[0116] In a specific embodiment, the biomarker molecule is an AGE, the abnormal form of the biomarker molecule is an stimulatory AGE, the binding molecule is sRAGE, the detectable binding agent is an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and the kit or device further comprises a competitor molecule of the biomarker molecule, which may be G-BSA or G-OVA.

[0117] In some embodiments, fluorescent nanoparticles may be provided as a detection reagent. The detection reagent may be provided separately from the device or kit of the present disclosure, or may be provided integrally with the device or kit of the present disclosure. The detection reagent may be mixed with the sample or may be included in the conjugate portion. When the detection reagent is mixed with the sample, the conjugate portion may be omitted. When the sample is developed horizontally, the membrane preferably has a conjugate portion containing the detection reagent. When the sample is developed vertically, the conjugate portion is preferably omitted, and the detection reagent is preferably premixed in the sample.

[0118] (composition) In another aspect, the present disclosure provides a composition for use in a device, system, or kit for detecting or quantifying an abnormal form of a biomarker molecule, comprising fluorescent nanoparticles, the device, system, or kit comprising a membrane that develops a sample by capillary action, the membrane comprising a detection section comprising a detection binding agent that specifically binds to the biomarker molecule or its competitor molecule, and a control section comprising a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with the abnormal form of the biomarker molecule or its competitor molecule, the fluorescent nanoparticles being used by labeling the binding molecule. In some embodiments, the device, system, or kit may comprise the sample contact section and the fluorescent nanoparticles as part of the membrane or as separate elements.

[0119] In a further aspect, the present disclosure provides a composition for use in a device, system, or kit for detecting or quantifying an aberrant form of a biomarker molecule, the composition comprising a binding molecule labeled with a fluorescent nanoparticle, the binding molecule being capable of forming a conjugate with the aberrant form of the biomarker molecule or its competitor molecule, the device, system, or kit comprising a membrane for developing a sample by capillary action, the membrane comprising a detection section comprising a detection binding agent that specifically binds to the biomarker molecule or its competitor molecule, and a control section comprising a control binding agent that specifically binds to the binding molecule. In some embodiments, the device, system, or kit may comprise the sample contact section and the fluorescent nanoparticles as part of the membrane or as separate elements.

[0120] (Modified LDL detection system) In one aspect, the present disclosure provides a system or device for detecting or quantifying denatured LDL, comprising a membrane that develops a sample by capillary action. The membrane comprises a conjugate portion containing CTLD14 labeled with fluorescent nanoparticles, a detection portion containing an anti-LDL antibody, an anti-denatured LDL antibody, or an anti-ApoB antibody or an antigen-binding fragment thereof, and a control portion containing a binding molecule for CTLD14. In some embodiments, the system or device may include the sample contact portion and the fluorescent nanoparticles as part of the membrane or as separate components. The system of the present disclosure is capable of detecting denatured LDL more easily and quickly than conventional methods. Furthermore, it is possible to detect denatured LDL using biological samples, such as blood samples (e.g., whole blood, serum, plasma), containing many impurities. The system of the present disclosure may be a lateral flow assay system. The system of the present disclosure is advantageous because it can detect denatured LDL in blood samples containing many impurities without requiring additional steps to remove the impurities.

[0121] In some embodiments, the conjugate moiety may be provided as a separate element from the membrane.

[0122] The sample contact portion of the present disclosure is a portion that can come into contact with a sample (e.g., blood, etc.), and may have any shape and be made of any material as long as it can be in contact with the sample, although materials that react with the sample should be avoided if possible. The sample contact portion may be provided as a part of the membrane or as a separate element, but in either case, it must be connected to the control portion and the detection portion so that the sample can penetrate therethrough by capillary action.

[0123] In some embodiments, the sample contact portion may include a blood cell separation filter. A blood cell separation filter refers to a filter that filters red blood cells, white blood cells, and platelets and sends components other than blood cells to a membrane. Examples of such filters include, but are not limited to, FUSION5, LF1, MF1, and VF2. In a preferred embodiment, the blood cell separation filter may be FUSION5 or LF1.

[0124] The conjugate moiety in the present disclosure comprises fluorescent nanoparticle-labeled CTLD 14. The conjugate moiety may be configured with a structure and material that allows contact between the fluorescent nanoparticle-labeled CTLD 14 and a sample.

[0125] In some embodiments, the fluorescent nanoparticles may be selected from compound semiconductor nanoparticles or fluorescent latex particles, preferably amorphous silica particles, and most preferably fluorescent silica nanoparticles (e.g., Quartz Dot) whose particle surfaces are covered with highly hydrophilic silanol groups, making them less susceptible to adsorption through hydrophobic interactions. The fluorescent nanoparticle-labeled CTLD14 is placed in the conjugate moiety. In some embodiments, CTLD14 is biotinylated, His-tagged, Myc-tagged, Flag-tagged, E-tagged, or Strep-tagged, and in each case, the binding molecule for CTLD14 may be streptavidin, anti-His antibody, anti-Myc antibody, anti-Flag antibody, anti-E-tag antibody, or Strep-Tactin.

[0126] In some embodiments, CTLD14 may have a silkworm-type glycan. CTLD14 with such a silkworm-type glycan may be produced by silkworms or may have an artificially added glycan, but production by silkworms is preferred. CTLD14 produced by silkworms incorporating a biotin ligase in a co-expressible manner can be biotinylated by including a biotinylation tag within the CTLD14 sequence. Examples of biotinylation tags include, but are not limited to, BioEase.tag, Avi.tag, and any sequence capable of being biotinylated. The use of biotinylated CTLD14 allows the use of streptavidin, which is relatively readily available, as a control site. The resulting biotinylated CTLD14 is stable and excellent even when conjugated with fluorescent nanoparticles under alkaline conditions. In certain embodiments, CTLD14 may have at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence of SEQ ID NO: 2. Methods for producing biotinylated CTLD14 having silkworm-type glycans are also described in detail in WO 2016 / 051808, which is incorporated herein by reference.

[0127] In the system or device of the present disclosure, the detection portion (test spot or line) contains an anti-denatured LDL antibody, an anti-LDL antibody, or an anti-ApoB antibody. The detection portion is a portion that enables detection of the presence of a target component (e.g., denatured LDL, irritating AGEs) in a sample via these antibodies. Therefore, the detection portion may be configured with any shape and material that enables detection.

[0128] In the system or device of the present disclosure, the control area (control spot or line) is a portion that confirms the development of a sample on the device and contains a binding molecule for CTLD14 (e.g., an antibody against CTLD14, or streptavidin that detects biotin if CTLD14 is biotinylated). In the detection area (test spot or line), the binding molecule for CTLD14 binds to CTLD14, and subsequently or in parallel interacts with the antibody, causing the fluorescent nanoparticle-labeled CTLD14 to aggregate and increase in fluorescence intensity, enabling detection or quantification of the target component. Therefore, the detection area may be configured with any shape and material that allows binding, control, and detection. Detection or quantification is also described in detail in (Detection or Quantification Method).

[0129] (Irritating AGEs detection system) In another aspect, the present disclosure provides a system for detecting or quantifying stimulatory AGEs, comprising a membrane that develops a sample by capillary action, the membrane comprising a conjugate portion comprising fluorescent nanoparticle-labeled sRAGE, a detection portion comprising an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and a control portion comprising a binding molecule for sRAGE. In some embodiments, the conjugate portion may further comprise G-BSA or G-OVA. In some embodiments, the conjugate portion may be provided as a separate element from the membrane.

[0130] Without wishing to be bound by theory, irritant AGEs in a sample can be detected by a decrease in the density (fluorescence intensity) of the spot on the test line due to competition for sRAGE. For example, in a system in which an anti-BSA antibody is spotted on the test line, fluorescent nanoparticle-labeled sRAGE and the sample are added to a reaction buffer containing glucose-modified BSA (an AGE with weak G-BSA activity). In the absence of irritant AGEs in the sample, the fluorescent nanoparticle-labeled sRAGE binds to G-BSA, is captured by the antibody, and agglutinates at the test line, emitting fluorescence. In contrast, in the presence of irritant AGEs in the sample, the amount of G-BSA binding to sRAGE decreases, resulting in less aggregation of the fluorescent nanoparticle-labeled sRAGE at the test line and a decrease in the fluorescence of the test spot. The presence of irritant AGEs can be detected by the degree of inhibition of the fluorescence intensity of the test line. When using an anti-G-BSA antibody on the test line, BSA cannot be used in the conjugate buffer or blocking buffer; therefore, casein, PVA, etc. can be used as appropriate. As another example, a system can be implemented in which anti-OVA (ovalbumin) antibody is used as the test line and G-OVA is used as the competing AGE.

[0131] The system or device of the present disclosure may preferably include a blood cell separation unit. The blood cell separation unit is desirable when the sample contains or is expected to contain blood cells or blood components. Since blood may contain components that inhibit detection, it may be advantageous to be able to separate blood cells in particular.

[0132] In a further aspect, the present disclosure provides a device or kit for detecting or quantifying an aberrant form of a biomarker molecule by conjugation with the biomarker molecule, the device comprising a membrane that develops a sample by capillary action. The membrane comprises a detection section containing a competitor molecule of the biomarker molecule and a control section containing a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with the aberrant form of the biomarker molecule or its competitor molecule. The kit or device comprises a sample contact section and fluorescent nanoparticles as part of the membrane or as a separate element. Binding of the binding molecule to the biomarker molecule in the sample can inhibit binding to the competitor molecule in the detection section, leading to a decrease in fluorescence intensity. The biomarker molecule in the sample can be quantified based on the decrease in fluorescence intensity. In another aspect, the present disclosure may also provide a composition comprising fluorescent nanoparticles that label the binding molecule or a binding molecule labeled with fluorescent nanoparticles for use in the above kit or device. A schematic diagram of a detection system comprising a competitor molecule in the detection section is shown in Figure 17.

[0133] In some embodiments, the biomarker molecule is an AGE, the abnormal form of the biomarker molecule is an stimulatory AGE, the binding molecule is sRAGE, and the competitor molecule can be non-glycated BSA, non-glycated OVA, CML-BSA, CML-OVA, G-BSA, or G-OVA. In a specific embodiment, the competitor molecule can be non-glycated BSA, non-glycated OVA, CML-BSA, or CML-OVA, preferably non-glycated BSA or CML-BSA, and most preferably non-glycated BSA or CML-BSA.

[0134] (Detection or quantification method) In another aspect, the present disclosure provides a method for detecting or quantifying an aberrant form of a biomarker molecule, the method comprising the steps of: providing a sample; mixing the sample with a binding molecule labeled with a fluorescent nanoparticle, the binding molecule being capable of forming a conjugate with the aberrant form of the biomarker molecule or a competitor molecule thereof; contacting the mixed sample with the sample contact portion of the membrane in the device or kit of the present disclosure; and, after the contact, adding a buffer solution as necessary.

[0135] In another aspect, the present disclosure provides a method for detecting or quantifying modified LDL or stimulatory AGEs, the method comprising the steps of providing a blood sample, mixing the blood sample with a buffer and an anticoagulant (e.g., heparin), contacting the mixed blood sample with a sample contact portion of a system or device of the present disclosure, and adding the buffer after contact.

[0136] In some embodiments, the buffer solution includes, but is not limited to, phosphate buffered saline (PBS). In a preferred embodiment, the buffer solution may be PBS(-). The buffer solution may or may not contain other proteins (which do not affect subsequent manipulations) such as albumin (e.g., bovine serum albumin (BSA)), casein, or PVA. In a specific embodiment, the buffer solution may be phosphate buffered saline (PBS)(-) supplemented with BSA.

[0137] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0138] (Example 1: Purification of single-chain antibodies from extracts of the middle silk gland of silkworm) We generated transgenic silkworms carrying three constructs: a USA-Biotin-tagged anti-LDL single-chain antibody (in which a BioEase-tagged anti-LDL single-chain antibody was inserted downstream of the target sequence USA); a USA-BirA construct (in which a biotin ligase (BirA) was inserted downstream of the target sequence USA); and a sericin 1 promoter (Ser1)-GAL construct that specifically expresses GAL4 in the middle silk gland. These silkworms express the Biotin-tagged anti-LDL single-chain antibody and BirA specifically in the middle silk gland. Furthermore, biotinylated single-chain antibodies can be produced by feeding fifth-instar larvae a diet containing 20 μg of biotin per gram, providing the necessary biotin for biotinylation of the anti-LDL single-chain antibody. In this example, non-biotinylated single-chain antibodies were used. The middle silk gland silk glands were removed immediately before cocoon formation, and proteins were extracted with Triton X-100 / PBS(-).

[0139] Sericin was removed from the middle silk gland extract by freeze-thawing, and the resulting lysate was added to TALON (Clontech) resin (a Co-based metal chelating affinity chromatography resin) equilibrated with PBS. Imidazole was added to a final concentration of 5 mM, and the mixture was slowly stirred for 2 hours. The resin was washed with PBS containing 10 mM imidazole, and the single-chain antibody was then eluted with PBS containing 1 M imidazole. The eluted fractions were collected and dialyzed against PBS(-). For immunochromatographic assays, the dialyzed sample was concentrated using a centrifugal ultrafiltration filter (Amicon Ultra, Merck), and the buffer was replaced with 10 mM phosphate buffer (pH 7.5).

[0140] (result) The results are shown in Figure 1. As shown, the single-chain antibody was successfully expressed in the middle silk gland expression system.

[0141] Example 2: Lateral Flow (Immunochromatographic) Assay (Preparation of fluorescent nanoparticle-labeled CTLD14) To perform an immunochromatographic assay for detecting oxidized LDL, CTLD14, the ligand recognition site of the LOX-1 receptor that recognizes oxidized LDL, was modified with silica nanoparticles (QuartzDot, Furukawa AE). The preparation method was performed according to the QuartzDot instruction manual, and is outlined below.

[0142] QuartzDots (100 nm particle size) were mixed in a tube with dimethylformamide containing N-(4-aminophenyl)maleimide (final concentration 2 mg / ml) to a final concentration of 2 mg / ml. The mixture was incubated at 30°C for 30 minutes, then centrifuged and the supernatant removed. MES buffer (pH 6.0, final concentration 100 mM), N-hydroxysuccinimide (final concentration 23 mg / ml), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (final concentration 2.88 mg / ml), and CTLD14 solution (final concentration 30 μg / ml) were added to the tube and ultrasonically dispersed. The mixture was then incubated at 30°C for 1 hour, centrifuged, and the supernatant removed. Blocking agents A and B and MilliQ water were added to the precipitate in a 1:2:7 ratio, ultrasonically dispersed, and then centrifuged. The supernatant was removed. The precipitate was ultrasonically dispersed in 10 mM phosphate buffer (pH 7.5), then centrifuged and the supernatant was removed. Phosphate buffer was added again and the mixture was ultrasonically dispersed to obtain fluorescent nanoparticle-labeled CTLD14.

[0143] (Preparation of immunochromatographic strips using single-chain antibodies) On a half-strip membrane consisting only of an absorbent pad and membrane, 0.5 μl of streptavidin was spotted as a control spot and 0.5 μl of single-chain antibody was spotted as a test spot, and then dried at 37°C for 1 hour. After drying, the spots were immersed in a 1% BSA / PBS solution at room temperature and gently shaken for 15 minutes, then gently washed twice with MilliQ water, and then gently shaken for another 5 minutes. After washing, the strips were gently patted dry and air-dried overnight on a paper towel covered with Kimwipes. A fluorescence detector is required for detection, but a simple detector is sufficient. In this example, a Typhoon fluorescence image analyzer was used.

[0144] (result) As shown in Figure 2, it was demonstrated that oxidized LDL can be detected using fluorescent silica nanoparticle-labeled CTLD14. Figure 3 shows the results of measuring the fluorescence intensity of each spot. It was revealed that the concentration of oxidized LDL can be quantitatively indicated based on the fluorescence intensity.

[0145] Modification with fluorescent silica nanoparticles must be performed in a salt-free buffer (PB, not PBS). Because silkworm MSG-derived CTLD14 maintained its function even in the absence of salt, modification with fluorescent silica nanoparticles was possible, and a lateral flow assay system capable of quantification by measuring fluorescence intensity was developed.

[0146] Example 3: Detection of added oxidized LDL by immunochromatographic assay (material and method) 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. 2 μl of fluorescent nanoparticle-labeled CTLD14 was added to the model sample in a microtube and mixed. The sample was then transferred to a well of a 96-well microplate, and the strip was inserted and spread vertically. After approximately 30 minutes of spreading, the strip was fully developed, and a Typhoon FLA9600 was used to capture fluorescent images and simultaneously measure the fluorescence intensity.

[0147] Next, 1000-fold or 5000-fold diluted serum to which oxidized LDL was added was used as a sample to examine whether serum components interfere with development.

[0148] (result) The results are shown in Figure 3. The upper figures in Figure 3B and C show the fluorescent images, and the lower figures show the fluorescence intensity (C: control spot, T: test spot). As shown, when the fluorescence intensity of each spot on the strip was measured after development, it became clear that it was possible to quantitatively indicate the concentration of oxidized LDL. The results using serum samples are shown in Figure 4. When undiluted serum was used, inhibition was observed, but when diluted 1000-fold or more, it was confirmed that there was no effect on detection.

[0149] Example 4: Comparison of single-chain antibody spots by immunochromatographic assay using serum from hyperlipidemic patients Based on the left diagram of Figure 6 (control target values ​​and classification of LDL, HDL, and TG for patients with dyslipidemia as described in the Guidelines for Prevention of Atherosclerotic Diseases), the LDL, HDL, and TG values ​​were classified into levels 0 to 4 depending on whether they were abnormal, borderline, or within the control target range. Furthermore, the correlation between each classification and oxidized LDL levels (measured in accordance with International Publication No. 2016 / 051808) was analyzed. Next, oxidized LDL in serum at each level was measured according to Example 2.

[0150] (result) The results are shown in Figure 7. The center image in Figure 7 shows the fluorescent image after development, and the right image shows the fluorescence intensity. Compared to "Serum 1," which was at level 0, the sample group containing items classified as abnormal (Serum 2-6) had stronger spot fluorescence intensity. This suggests that the simple and rapid quantitative value of oxidized LDL concentration can be used to determine the patient's condition.

[0151] Example 5: Detection of oxidized LDL in whole blood (material and method) Oxidized LDL and fluorescent nanoparticle-labeled CTLD14 were added to a microtube containing whole blood and mixed, then transferred to a well of a microplate. A full strip with a blood cell removal filter (single-chain antibodies of different concentrations were applied to the test spots, and streptavidin was applied to the control spots) was inserted and allowed to spread vertically.

[0152] (result) As shown in the right image of Figure 8, the fluorescence image acquired after development confirmed a significant increase in the fluorescence intensity of the test spot in the sample to which oxidized LDL had been added, demonstrating that simple and rapid detection and quantification of oxidized LDL in whole blood is possible.

[0153] Example 6: Preparation of fluorescent nanoparticle-labeled sRAGE for AGE detection using silica nanoparticles The preparation method for fluorescent nanoparticle-labeled sRAGE using silica nanoparticles (QuartzDot, Furukawa AE) was performed according to the QuartzDot instruction manual with some modifications. The outline is as follows.

[0154] QuartzDot (100 nm particle size) was mixed in a tube with dimethylformamide containing N-(4-aminophenyl)maleimide (final concentration 2 mg / ml) to a final concentration of 2 mg / ml, incubated at 30°C for 30 minutes, centrifuged, and the supernatant was removed. MES buffer (pH 6.0, final concentration 100 mM), N-hydroxysuccinimide (final concentration 23 mg / ml), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (final concentration 2.88 mg / ml), and sRAGE solution (final concentration 30 μg / ml) were added to the tube and dispersed ultrasonically. The mixture was then incubated at 30°C for 1 hour, centrifuged, and the supernatant was removed. The precipitate was blocked using a 1:2:7 mixture of QuartzDot's included blocking agents A and B and MilliQ water, or phosphate buffer or Tris buffer containing 0.05-1.0% PVA, or phosphate buffer or Tris buffer containing 0.05-2.5% casein. The blocking agent was added, the mixture was ultrasonically dispersed, and then centrifuged to remove the supernatant. The precipitate was then ultrasonically dispersed in 10 mM phosphate buffer (pH 7.5), centrifuged, and the supernatant was removed. Phosphate buffer was added again, and the mixture was ultrasonically dispersed to produce fluorescent nanoparticle-labeled sRAGE.

[0155] Fluorescent nanoparticle-labeled sRAGE was added to a microtube containing whole blood and mixed, then transferred to a well of a microplate. A full strip (no antibody applied to the test spots, streptavidin applied to the control spots) with a blood cell removal filter was inserted and allowed to spread vertically.

[0156] (result) The results are shown in Figure 9. Fluorescence images obtained after development confirmed that fluorescent nanoparticle-labeled sRAGE could be developed on the full strip without being affected by whole blood.

[0157] (Example 7: Detection of AGEs by lateral flow detection method using fluorescent nanoparticles) Preparation of AGE detection reagent using fluorescent nanoparticles (silica nanoparticle-labeled sRAGE) The fluorescent nanoparticle (QuartzDot, Furukawa Electric Advanced Engineering)-labeled sRAGE was prepared according to the QuartzDot instruction manual with some modifications. The outline of the procedure is as follows:

[0158] QuartzDots (100 nm particle size) were mixed in a tube with dimethylformamide containing N-(4-aminophenyl)maleimide (final concentration 2 mg / ml) to a final concentration of 2 mg / ml, incubated at 30°C for 30 minutes, centrifuged, and the supernatant removed. MES buffer (pH 6.0, final concentration 100 mM), N-hydroxysuccinimide (final concentration 23 mg / ml), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (final concentration 2.88 mg / ml), and sRAGE solution (final concentration 30-50 μg / ml) were added to the tube and dispersed ultrasonically. The mixture was then incubated at 30°C for 1 hour, centrifuged, and the supernatant removed. The precipitate was blocked using a 1:2:7 mixture of QuartzDot's included blocking agents A and B with MilliQ water, or phosphate buffer containing 0.1-0.5% PVA, or phosphate buffer containing 0.5-1.0% casein. The blocking agent was added, and the mixture was ultrasonically dispersed, followed by centrifugation and removal of the supernatant. The precipitate was then ultrasonically dispersed in 10 mM phosphate buffer (pH 7.5), centrifuged, and the supernatant was removed. Phosphate buffer was added again, and the mixture was ultrasonically dispersed to produce fluorescent nanoparticle-labeled sRAGE.

[0159] Preparation of lateral flow strips 0.5 μl of a control spot sample (streptavidin) and a test spot sample (CML-AGE-BSA, anti-AGE antibody, etc.) were spotted at appropriate locations on the membrane of a full strip consisting of an absorbent pad, membrane, and sample pad (blood cell separation pad), or a half strip consisting of only an absorbent pad and membrane, and then dried at 37°C for 30 minutes to 2 hours. For blocking, the spots were dried and then immersed in a blocking buffer (e.g., 0.5-1.0% BSA, 0-0.05% Tween-20 in 0.05-0.1% casein / PBS, 0-0.05% Tween-20 in 0.05-0.1% PVA / PBS, 0.05-0.1% casein / TBS, or 0-0.1% casein in 1x, 1 / 2x, or 1 / 5x Pierce Protein-free (PBS / TBS) Blocking buffer) at room temperature for 15 minutes with gentle shaking. Then, the spots were gently washed twice with MilliQ water, followed by another 5 minutes with gentle shaking. After washing, the spots were gently patted dry and air-dried overnight on a paper towel covered with Kimwipes.

[0160] Detection of AGEs by lateral flow detection method 10 ng–4 μg of AGE solution (CML-AGE-BSA, Glucose-AGE-BSA, Glyceraldehyde-AGE-BSA, Glycolaldehyde-AGE-BSA, etc.) was added to 60 μl of reaction buffer (0.5–1.0% BSA, 0–0.05% Tween-20 in 0.05–0.1% casein / PBS, 0–0.05% Tween-20 in 0.05–0.1% PVA / PBS, 0.05–0.1% casein / TBS, 0–0.1% casein in 1×, 1 / 2×, or 1 / 5× Pierce Protein-free (PBS / TBS) Blocking buffer, etc.) and used as a model sample. For samples containing serum, frozen serum was thawed on ice and centrifuged at 1,000 × g for 15 minutes. The supernatant was diluted 500-fold or 1,000-fold with PBS, and 60 μl of the diluted serum was used. The sample was mixed with the detection reagent (QuartzDot-labeled sRAGE). The mixture was transferred to a microplate well, and a prepared lateral flow half-strip was inserted. After 20 to 30 minutes, when the mixture in the well was completely absorbed by the strip, the fluorescence intensity of the test and control spots was measured using a Typhoon FLA 9500. For samples containing whole blood, 75 μl of blood diluted 500-fold with reaction buffer was added to the AGE solution and mixed with the detection reagent (QuartzDot-labeled sRAGE). The mixture was then transferred to a microplate well, and a full strip with a blood cell separation pad attached was inserted into the sample pad. After 5 to 10 minutes, when the strip had finished absorbing the mixture, 50 μl of reaction buffer was added to the wells. After another 30 minutes or so, when all the solution had been absorbed, the fluorescence intensity of the test and control spots was confirmed using a Typhoon FLA 9500.

[0161] Model sample, AGE / PBS(-) An anti-AGE antibody was applied to the test spot, and the sample (unglycated BSA or glucose-glycated BSA) was added to the buffer solution and developed. AGEs bound to fluorescent nanoparticle-labeled biotinylated sRAGE bound to the test line, resulting in an increase in fluorescence intensity (image acquired with an image analyzer) (Figure 11A). Furthermore, CML-glycated BSA (CML-BSA, antagonistic AGEs) was applied to the test spot, and the sample (unglycated BSA or CML-BSA) was added to the buffer solution and developed. Binding of fluorescent nanoparticle-labeled biotinylated sRAGE to the test line was inhibited by AGEs, resulting in a decrease in fluorescence intensity (image acquired with an image analyzer) (Figure 11B). Figure 17 shows an overview of the detection system in which CML-BSA was applied to the test spot.

[0162] Detection of AGEs in serum An anti-AGE antibody was applied to the test spot, and human serum samples (unglycated BSA or glucose-glycated BSA) were added and developed. AGEs bound to fluorescent nanoparticle-labeled biotinylated sRAGE bound to the test line, resulting in an increase in fluorescence intensity (Figure 12A). Furthermore, CML-BSA (antagonistic AGEs) was applied to the test spot, and samples (unglycated BSA or CML BSA) were added to the buffer solution and developed. AGEs inhibited the binding of fluorescent nanoparticle-labeled biotinylated sRAGE to the test line, resulting in a decrease in fluorescence intensity that depended on the AGE concentration (Figure 12B).

[0163] Detection of AGEs in whole blood An anti-AGE antibody was applied to the test spot, and fresh human blood samples (unglycated BSA or glucose-glycated BSA) were added and developed. AGEs bound to fluorescent nanoparticle-labeled biotinylated sRAGE bound to the test line, resulting in an increase in fluorescence intensity (Figure 13A). Furthermore, CML-BSA (antagonistic AGEs) was applied to the test spot, and fresh human blood samples (unglycated BSA or CML-BSA) were added and developed. Binding of fluorescent nanoparticle-labeled biotinylated sRAGE to the test line was inhibited by AGEs, resulting in a decrease in fluorescence intensity (Figure 13B).

[0164] Detection of AGEs in whole blood (example of use as a system to evaluate an individual's poor health using a single drop of blood) CML-BSA (antagonistic AGEs) was applied to a test spot, and a drop of blood was collected from the finger of two healthy individuals using a lancet, once at a normal time and once after about a week of feeling unwell due to overwork. The sample (unglycated BSA or CML-BSA) was added to the fresh blood immediately after collection and developed, and the degree of decrease in fluorescence intensity was compared. It was confirmed that the degree of decrease in fluorescence intensity was smaller when people were in poor health (Figure 14). This is thought to be because the concentration of AGEs in the blood increases due to AGEs, which are expected to accumulate when people are in poor health, making it difficult to see the inhibitory effect of the added AGEs (CML-BSA).

[0165] Detection of AGEs in serum of NASH patients Nonalcoholic fatty liver disease (NAFLD), a liver disease caused by obesity and lifestyle-related diseases, is rapidly increasing in Japan due to the increase in the obese population, and is becoming a national disease affecting 20-40% of adults. NAFLD is divided into nonalcoholic fatty liver (NAFL), which rarely progresses, and chronic, progressive nonalcoholic steatohepatitis (NASH), which can progress to liver cirrhosis and hepatocellular carcinoma. It is extremely important to distinguish between NAFLD and progressive NASH. An anti-AGE antibody was applied to the test spot, and human serum was added and developed. AGEs in the serum bound to the fluorescent nanoparticle-labeled biotinylated sRAGE, resulting in agglutination on the test line, and the AGE concentration could be measured as an increase in fluorescence intensity (Figure 15A). Furthermore, CML-treated BSA (antagonizing AGEs) was applied to the test spot, and human serum was added and developed. AGEs in the serum bound to the fluorescent nanoparticle-labeled biotinylated sRAGE, preventing agglutination on the test line, and the AGE concentration was measured as a decrease in fluorescence intensity (Figure 15B). A decrease in fluorescence intensity was confirmed in the patient serum group.

[0166] Detection of AGEs in the serum of patients with diabetic complications An anti-AGE antibody was applied to the test spot, and human serum was added and developed. AGEs in the serum bound to the fluorescent nanoparticle-labeled biotinylated sRAGE, resulting in aggregation on the test line. The AGE concentration was measured as an increase in fluorescence intensity (Figure 16). A correlation was observed between the increase in HbA1c value and the fluorescence intensity.

[0167] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but the present disclosure should not be construed as being limited to these embodiments. It is understood that the scope of the present disclosure should be interpreted solely by the scope of the claims. It is understood that a person skilled in the art can implement an equivalent scope based on the description of the present disclosure and common general technical knowledge from the description of specific preferred embodiments of the present disclosure. It is understood that the contents of the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification as if the contents themselves were specifically set forth in this specification. This application claims the benefit of priority from Japanese Patent Application No. 2019-203443, filed on November 8, 2019, the contents of which are incorporated by reference into this specification. [Industrial Applicability]

[0168] The present disclosure is useful in the field of disease diagnosis or predictive diagnosis industry. [Sequence List Free Text]

[0169] SEQ ID NO: 1: Nucleic acid sequence encoding CTLD14 SEQ ID NO: 2: Amino acid sequence encoding CTLD14 SEQ ID NO: 3: Nucleic acid sequence of RAGE SEQ ID NO: 4: Amino acid sequence of RAGE SEQ ID NO: 5: Nucleic acid sequence of sRAGE 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 chain antibody used in the present invention

Claims

1. A device or kit for detecting or quantifying an abnormal form of a biomarker molecule by forming a conjugate with the biomarker molecule, comprising a membrane that develops a sample by capillary action, the membrane comprising: a detection unit containing a detection binding agent that specifically binds to a biomarker molecule or its competitor molecule; a control section containing a control binding agent that specifically binds to a binding molecule capable of forming a conjugate with an abnormal form of the biomarker molecule or its competitor molecule; Including, The kit or device comprises a sample contact portion and fluorescent nanoparticles as part of a membrane or as separate elements, wherein the biomarker molecules are AGEs, the abnormal forms of the biomarker molecules are stimulatory AGEs, the binding molecule is sRAGE, the detection binding agent is an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and the kit or device further comprises a competitor molecule for the biomarker molecule, wherein the competitor molecule is G-BSA or G-OVA.

2. The kit or device according to claim 1 , wherein the membrane comprises, from upstream to downstream, the detection section and the control section, in this order.

3. 3. The kit or device according to claim 1, wherein the sample contact portion, the detection portion, and the control portion are arranged or connected to each other so that the sample penetrates into each other by capillary action.

4. The device or kit of any one of claims 1 to 3, wherein the fluorescent nanoparticles are provided as detection reagents.

5. The device or kit according to any one of claims 1 to 4, wherein the fluorescent nanoparticles are provided in the membrane as a sample mixture.

6. The device or kit according to any one of claims 1 to 5, wherein the sample contact section includes a blood cell separation section.

7. The device or kit according to claim 6 , wherein the blood cell separation unit is selected from FUSION 5, LF1, MF1, and VF2.

8. The device or kit according to any one of claims 1 to 7, wherein the sample is a blood sample.

9. The device or kit of claim 8 , wherein the blood sample is serum or whole blood.

10. 1. A method for detecting or quantifying an aberrant form of a biomarker molecule, comprising: Providing a sample removed from the body; mixing the sample with a binding molecule labeled with a fluorescent nanoparticle, the binding molecule capable of forming a conjugate with an aberrant form of a biomarker molecule or a competitor thereof; The sample contact portion in the device or kit according to any one of claims 1 to 9. contacting the mixed sample with After contacting, adding a buffer solution; A method comprising:

11. 1. A composition for use in a device, system, or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising a binding molecule labeled with a fluorescent nanoparticle, the binding molecule being capable of forming a conjugate with the aberrant form of the biomarker molecule or a competitor molecule thereof; The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: Sample contact area and a detection unit containing a detection binding agent that specifically binds to a biomarker molecule or its competitor molecule; a control section containing a control binding agent that specifically binds to the binding molecule; Including, A composition, wherein the biomarker molecule is an AGE, an abnormal form of the biomarker molecule is an stimulatory AGE, the binding molecule is sRAGE, the detectable binding agent is an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and the device, system, or kit further comprises a competitor molecule of the biomarker molecule, wherein the competitor molecule is G-BSA or G-OVA.

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