Visual detection of modified LDL or irritating AGEs

Abstract

The present disclosure provides a technique for simply, rapidly, and visually detecting modified LDL or stimulating AGEs. The present disclosure also provides a device or kit for detecting or quantifying an abnormal type of a biomarker molecule by forming a conjugate with the biomarker molecule, the device or kit comprising a membrane for developing a sample by utilizing a capillary action, wherein the membrane comprises: a detection part including a binding agent for detection which binds specifically to the biomarker molecule or a competitor molecule thereof; and a control part including a binding agent for control which binds specifically to a binding molecule having conjugate-forming ability with the abnormal type of the biomarker molecule or a competitor molecule thereof, and the kit or device comprises a sample contact part and a metal colloid as portions of the membrane or separate elements.

Description

[Technical Field]

[0001] The present invention relates to a technique for easily and quickly visually detecting denatured 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 population of molecules with varying degrees of modification. The present inventors previously succeeded in developing a method for comprehensively detecting only oxidized LDL (the true bad cholesterol), which triggers disease in vivo, 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 evaluating the effects of lifestyle modifications, such as exercise and dietary habits, on the suppression of disease progression, and the status of disease management through medication, etc.

[0003] AGEs (Advanced glycation end products) are the products of protein glycation, but are a general term for a variety of structures. In recent years, it has been discovered that AGEs, which are generated by glycative stress in the human body, also exist, and that these include irritating AGEs that can induce diabetic complications and age-related diseases (rheumatoid arthritis, Alzheimer's disease, etc.). However, like the oxidized LDL detection method, the developed method is intended for use in research and testing institutions, and there is a need for the development of a simple and rapid method for evaluating the effectiveness of lifestyle improvements such as exercise and diet in inhibiting disease progression, as well as 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 detection system, such as a lateral flow assay (immunochromatography), that can detect oxidized LDL or irritating AGEs. Specifically, the detection system uses a recognition element modified with a metal colloid that recognizes oxidized LDL or irritating AGEs, enabling simple and rapid detection 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 a metal colloid as part of a membrane or as a separate element. (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) 3. The kit or device according to item 1 or 2, 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. (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) 10. The device or kit according to any one of items 1 to 9, 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. (Item 11) 11. The device or kit according to any one of items 1 to 10, wherein the metal colloid is provided as a detection reagent. (Item 12) 11. The device or kit according to any one of items 1 to 10, wherein the metal colloid is 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) 15. The device or kit according to any one of items 1 to 14, wherein the metal colloid is selected from the group consisting of gold, silver, platinum, and palladium. (Item 16) Item 16. The device or kit according to item 15, wherein the metal colloid is platinum colloid. (Item 17) 7. The device or kit of item 6, 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 18) 7. The device or kit according to item 6, wherein the CTLD14 has a silkworm-type glycan. (Item 19) 7. The device or kit of item 6, wherein the CTLD14 is biotinylated and the control binding agent is streptavidin. (Item 20) 20. The device or kit according to any one of items 1 to 19, wherein the sample is a blood sample. (Item 21) 21. The device or kit according to item 20, wherein the blood sample is serum or whole blood. (Item 22) 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 metal colloid, the binding molecule being capable of forming a conjugate with an aberrant form of a biomarker molecule or a competitor molecule thereof; A step of contacting the mixed sample with the sample contact portion of the device or kit according to any one of items 1 to 20; After contacting, adding a buffer solution as needed; A method comprising: (Item 23) 1. A composition for use in a device, system or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising a metal colloid, The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: 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 metal colloid is used by labeling the binding molecule. composition. (Item 24) 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 metal colloid, 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: 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 25) 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 a metal colloid as part of a membrane or as a separate element. (Item 26) 26. The kit or device of claim 25, having one or more of the features described above. (Item 27) 1. A composition for use in a device, system or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising a metal colloid, The device, system, or kit includes a membrane that develops a sample by capillary action, and the membrane comprises: 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 metal colloid is used by labeling the binding molecule. composition. (Item 28) A composition for use in a device, system, or kit for detecting or quantifying an abnormal form of a biomarker molecule, comprising a binding molecule labeled with a metal colloid, wherein the binding molecule has the ability to form a conjugate with the abnormal 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: 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 29) 29. The composition of claim 27 or 28, characterized by one or more of the above items. (Item 30) 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 metal colloid, the binding molecule being capable of forming a conjugate with an aberrant form of a biomarker molecule or a competitor molecule thereof; Contacting the mixed sample with the sample contact portion of the device or kit according to Item 25; After contacting, adding a buffer solution as needed; A method comprising: (Item 31) 31. The method of claim 30, 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 a metal colloid 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 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 molecules, 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 metal colloid is provided as a detection reagent. (Item 10A) The device or kit according to any one of items 1A to 8A, wherein the metal colloid is 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) The device or kit according to any one of Items 1A to 12A, wherein the metal colloid is selected from the group consisting of gold, silver, platinum, and palladium. (Item 14A) The device or kit according to item 13A, wherein the metal colloid is platinum colloid. (Item 15A) 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 16A) The device or kit according to Item 4A, wherein the CTLD14 has a silkworm-type glycan. (Item 17A) The device or kit of item 4A, wherein the CTLD14 is biotinylated and the control binding agent is streptavidin. (Item 18A) The device or kit according to any one of items 1A to 17A, wherein the sample is a blood sample. (Item 19A) 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 metal colloid, the binding molecule being capable of forming a conjugate with an aberrant form of a biomarker molecule or a competitor molecule 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 18A; After contacting, adding a buffer solution as needed; A method comprising: (Item 20A) 1. A composition for use in a device, system or kit for detecting or quantifying an aberrant form of a biomarker molecule, comprising a metal colloid, 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 metal colloid is used by labeling the binding molecule. composition. (Item 21A) 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 metal colloid, 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.

[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] According to the present disclosure, a detection system capable of easily and quickly detecting disease-related oxidized LDL or irritating AGEs is provided. 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 treatment, 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 electrophoretic images confirming the expression of CTLD14 using the middle silk gland and posterior silk gland expression systems. [Figure 2] Figure 2 shows the detection of LDLs by sandwich ELISA using CTLD14 derived from the middle silk gland (MSG) and posterior silk gland (PSG) and chicken anti-LDL polyclonal antibody. Open squares represent native LDL, filled triangles represent AGE-LDL, open circles represent partially oxidized LDL, and filled circles represent fully oxidized LDL. [Figure 3] FIG. 3 shows the preparation of a standard curve for oxidized LDL by sandwich ELISA using middle silk gland (MSG)-derived CTLD14 and chicken anti-LDL polyclonal antibody, and the calculation of the amount of oxidized LDL in human serum. [Figure 4] Figure 4 shows an overview of the modification of CTLD14 with metal colloids and a schematic diagram of the half-strips. [Figure 5] FIG. 5 shows a schematic diagram of the principle of detection of modified LDL by lateral flow assay. [Figure 6] FIG. 6 shows the results of measuring the circular dichroism (CD) spectrum of MSG-derived CTLD14. [Figure 7] FIG. 7 shows the results of a lateral flow assay using a single-chain antibody at the test line. [Figure 8] Figure 8 shows the results of the lateral flow assay when chicken polyclonal antibodies were used in the test line. [Figure 9]FIG. 9 shows the results of a lateral flow assay using an anti-ApoB monoclonal antibody as the test line. [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 an outline of the whole blood sample preparation method and the results of the effectiveness of metal colloids using Full Strip. [Figure 12] FIG. 12 shows the results of detecting irritating AGEs using half strips. [Figure 13] FIG. 13 shows the results of verifying the effectiveness of metal colloid-labeled sRAGE using full strip. [Figure 14] FIG. 14 shows the results of the detection of AGEs in whole blood by lateral flow assay. [Figure 15] FIG. 15 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, the term "kit" refers to a unit in which the components to be provided (e.g., membranes, devices, reagents, etc.) are provided, usually separated into two or more compartments. When multiple reagents or devices are provided independently, it may be convenient to provide them as a kit. Such kits preferably advantageously include instructions or manuals describing how to use the provided components (e.g., membranes or devices) or how to use the reagents.

[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 "metal colloid" is used in the same sense as commonly used in the art, and refers to a colloid in which metal particles (usually nano-sized, and may be extremely fine particles of about 0.25 to 0.001 micrometers) are dispersed in a fluid. A colloid or colloidal dispersion is a material state composed of two phases, one of which forms tiny droplets or particles (dispersed phase) and is dispersed in the other. When the dispersion medium is liquid, it is also called a colloidal solution. Specific examples include foams, emulsions, gels, and suspensions. Representative examples of metal colloids used herein include, but are not limited to, platinum group colloids such as gold colloid, silver colloid, platinum colloid, and palladium colloid. Platinum colloid may be preferred, but the present disclosure is not limited thereto.

[0019] 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.

[0020] 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).

[0021] 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.

[0022] 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.

[0023] 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 conjugating 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 conjugating 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 with the binding molecule capable of conjugating 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 with the competing molecule (e.g., G-BSA 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.

[0024] 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.

[0025] 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.

[0026] 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 the 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.

[0027] 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.).

[0028] 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).

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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, but is not limited to, positions 22 to 332 of SEQ ID NO: 6 or SEQ ID NO: 4.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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 (preferably 60% or more homology, more preferably 80% or more, 85% or more, 90% or more, or 95% or more homology) with a given gene at the amino acid or nucleotide level. 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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).

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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)).

[0059] 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).

[0060] 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.

[0061] 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."

[0062] 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).

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.).

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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."

[0084] 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.

[0085] 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.

[0086] 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).

[0087] 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)).

[0088] 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).

[0089] 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.

[0090] 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.

[0091] (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.

[0092] (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 a metal colloid as part of the membrane or as a separate element.

[0093] In some embodiments, the membrane may include, from upstream to downstream, a detection portion and a control portion, in that order.

[0094] 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.

[0095] 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.

[0096] In some embodiments, the binding molecule may be CTLD14 or sRAGE.

[0097] 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.

[0098] In some embodiments, the kits or devices of the present disclosure may or may not further include a competitor molecule of the biomarker molecule.

[0099] 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.

[0100] 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.

[0101] In some embodiments, the metal colloid 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 before use, 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 mixed in the sample in advance. The conjugate portion may be provided as a separate element from the membrane.

[0102] (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 a metal colloid, 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 metal colloid being used by labeling the binding molecule. In some embodiments, the device, system, or kit may comprise the sample contact section and the metal colloid as part of the membrane or as separate elements.

[0103] 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, comprising a binding molecule labeled with a metal colloid, wherein the binding molecule is capable of forming a conjugate with the aberrant form of the biomarker molecule or its competitor molecule, and the device, system, or kit comprises a membrane that allows sample development 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 metal colloid as part of the membrane or as separate elements.

[0104] (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 colloidal metal-labeled CTLD14, 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 a sample contact portion and 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.

[0105] In some embodiments, the conjugate moiety may be provided as a separate element from the membrane.

[0106] In the methods of the present disclosure, modified LDL can be quantified by colorimetric determination of coloration using techniques well known in the art, for example, the color density measurement function of a densitometer.

[0107] 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.

[0108] 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.

[0109] The conjugate moiety in the present disclosure comprises a metal colloid-labeled CTLD 14. The conjugate moiety may be composed of a structure and material that allows for conjugation of CTLD 14 with a sample upon contact.

[0110] In some embodiments, the metal colloid may be selected from the group consisting of colloidal gold, colloidal silver, colloidal platinum, and colloidal palladium, preferably the metal colloid is colloidal platinum or colloidal palladium, and most preferably the metal colloid is colloidal platinum. The metal colloid is disposed in a conjugate.

[0111] 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 can be streptavidin, an anti-His antibody, an anti-Myc antibody, an anti-Flag antibody, an anti-E tag antibody, or Strep-Tactin.

[0112] In some embodiments, CTLD14 may have a silkworm-type sugar chain. CTLD14 with such a silkworm-type sugar chain may be produced by silkworms or may be artificially glycosylated, 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 easily available, as a control site. The resulting biotinylated CTLD14 is stable and excellent even when conjugated with metal colloids 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. A method for producing biotinylated CTLD14 having a silkworm-type glycan is described in detail below. Reference may also be made to WO 2016 / 051808, which is incorporated herein by reference.

[0113] In the system or device of the present disclosure, the detection portion (test spot or line) contains an anti-LDL antibody, an anti-denatured 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.

[0114] 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 metal colloid-labeled CTLD14 to aggregate and develop color due to the plasmon effect, thereby 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).

[0115] (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 containing colloidal metal-labeled sRAGE, a detection portion containing an anti-BSA antibody or an anti-OVA antibody or an antigen-binding fragment thereof, and a control portion containing a molecule that binds to sRAGE. In some embodiments, the conjugate portion may further contain G-BSA as a competitor molecule. In some embodiments, the system may comprise a sample contact portion and fluorescent nanoparticles as part of the membrane or as separate components. In some embodiments, the conjugate portion may be provided as a separate component from the membrane.

[0116] Without wishing to be bound by theory, irritant AGEs in a sample can be detected by a decrease in the intensity (color) 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, metal colloid-labeled sRAGE and the sample are added to a reaction buffer containing glucose-modified BSA (a weakly active AGE). In the absence of irritant AGEs in the sample, metal colloid-labeled sRAGE binds to G-BSA, is captured by the antibody, and agglutinates at the test line, resulting in color development. However, in the presence of irritant AGEs in the sample, the amount of G-BSA binding to sRAGE decreases, resulting in less aggregation of metal colloid-labeled sRAGE at the test line, resulting in a decrease in color development at the test spot. The presence of irritant AGEs can be detected by the degree of inhibition of color development at the test line. When using an anti-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.

[0117] 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.

[0118] 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 a metal colloid as part of the membrane or as a separate element. Binding of the binding molecule to the competitor molecule in the detection section can inhibit binding to the competitor molecule in the detection section, leading to a decrease in color intensity. The biomarker molecule in the sample can be quantified based on the decrease in color intensity. In another aspect, the present disclosure may also provide a composition comprising a metal colloid that labels a binding molecule or a binding molecule labeled with a metal colloid 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 15.

[0119] In some embodiments, the biomarker molecule is AGEs, the abnormal form of the biomarker molecule is stimulatory AGEs, the binding molecule is sRAGE, and the competitor molecule can be non-glycated BSA, non-glycated OVA, CML-BSA, CML-OVA, G-BSA, G-OVA, F-BSA, or F-OVA. In certain embodiments, the competitor molecule can be G-BSA, G-OVA, F-BSA, or F-OVA, preferably G-BSA or F-BSA.

[0120] (Detection or quantification method) In another aspect, the present disclosure provides a method for detecting or quantifying an abnormal form of a biomarker molecule, the method comprising the steps of providing a sample, mixing the sample with a binding molecule labeled with a metal colloid, wherein the binding molecule is capable of forming a conjugate with the abnormal form of the biomarker molecule or a competitor molecule thereof, contacting the mixed sample with the sample contact portion in the device or kit of the present disclosure, and adding a buffer solution, if necessary, after the contact.

[0121] 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.

[0122] 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.

[0123] 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]

[0124] (Example 1: Purification of CTLD14 from extract of middle or posterior silk gland of silkworm) (material and method) We generated transgenic silkworms carrying three constructs: USA-Biotin-tagged CTLD14 (or USA-Biotin-tagged sRAGE), in which BioEase-tagged CTLD14 (or BioEase-tagged sRAGE) was inserted downstream of the target sequence USA; USA-BirA, in which biotin ligase (BirA) was inserted downstream of the target sequence USA; and sericin 1 promoter (Ser1)-GAL, which expresses GAL4 specifically in the middle or posterior silk gland. These silkworms express Biotin-tagged CTLD14 (or Biotin-tagged sRAGE) in a middle or posterior silk gland-specific manner. The 5th instar larvae express CTLD14 (sRAGE) and BirA. Furthermore, 20 μg of biotin per gram of diet was added to the diet to provide the biotin required for biotinylation of CTLD14 or sRAGE. The middle or posterior silk gland was removed immediately before cocoon formation, and proteins were extracted using TritonX-100 / PBS(-).

[0125] Sericin was removed from the middle or posterior silk gland extract by freeze-thawing, and the resulting lysate was bound to TALON resin (a Co-based metal chelating affinity chromatography resin) equilibrated with PBS. The resin was then washed with PBS containing 10 mM imidazole, and CTLD14 was eluted by gradually increasing the imidazole concentration in PBS. The 50 mM to 500 mM imidazole elution fractions were collected and dialyzed against PBS(-). The fractions were then bound to Mutein Matrix equilibrated with PBS, and eluted with PBS containing 1.5 mM to 3 mM biotin. The biotinylated CTLD14 was purified by PBS dialysis.

[0126] (result) The results are shown in Figure 1. As shown, biotinylated CTLD14 was successfully expressed in both the middle and posterior silk gland expression systems. Furthermore, the posterior silk gland expression system expressed biotinylated CTLD14 more efficiently than the middle silk gland expression system.

[0127] (Example 2: Detection of LDL ligands by sandwich ELISA using CTLD14 derived from the middle and posterior silk glands of silkworms and chicken anti-LDL polyclonal antibody) The detection of various LDL ligands was evaluated by sandwich ELISA using CTLD14 derived from the middle and posterior silk glands of Bombyx mori and chicken anti-LDL polyclonal antibody.

[0128] (material and method) CTLD14 (20 μg / ml) derived from the middle and posterior silk glands of Bombyx mori was dispensed into microplate wells in 50 μl aliquots and coated overnight at 4°C. The CTLD14 solution was discarded, and the wells were washed three times with 200 μl of PBS. 250 μl of 0.25% BSA / PBS was added and blocked for 2 hours at 25°C. The wells were then washed three times with 200 μl of PBS. Next, 100 μl of 0-4 μg / ml solutions of fully oxidized LDL (f-OxLDL), partially oxidized LDL (m-OxLDL), AGE-LDL, and native LDL (LDL) were added as ligands. After incubation for 2 hours at 25°C, the wells were washed five times with 200 μl of PBS, and 100 μl of anti-LDL chicken polyclonal antibody (diluted 1:6000 in 0.25% BSA / PBS) was added per well. After incubation at 25°C for 1 hour, the wells were washed five times with 200 μl of PBS, and 100 μl of anti-chicken IgY-HRP conjugate (diluted 1:6000 in 0.25% BSA / PBS) was added per well. After incubation at 25°C for 1 hour, the wells were washed five times with 200 μl of PBS, and 50 μl of TMB solution was added per well to confirm color development. The reaction was stopped by adding 50 μl of 1N HCl per well, and the absorbance at 450 nm was measured using a microplate reader.

[0129] (result) The results are shown in Figure 2. The ligands used were fully oxidized LDL (f-OxLDL), partially oxidized LDL (m-OxLDL), AGE-LDL, and native LDL (LDL). Open squares represent native LDL, filled triangles represent AGE-LDL, open circles represent partially oxidized LDL, and filled circles represent fully oxidized LDL. Left: Detection of LDL and modified LDL by sandwich assay using CTLD14 derived from the middle silk gland and anti-LDL chicken polyclonal antibody. Right: Detection of LDL and modified LDL by sandwich assay using CTLD14 derived from the posterior silk gland and anti-LDL chicken polyclonal antibody. Both CTLD14 derived from the middle and posterior silk glands recognized fully oxidized LDL well, and both CTLD14 derived from the middle and posterior silk glands had similar recognition abilities for other LDL ligands.

[0130] (Example 3: Preparation of a standard curve for oxidized LDL by sandwich ELISA using CTLD14 and chicken anti-LDL polyclonal antibody and calculation of the amount of oxidized LDL in human serum) (material and method) To remove the BioEase tag from CTLD14 derived from the middle silk gland of Bombyx mori, the antibody was treated with enterokinase and purified using HisTag on TALON resin to remove the cleaved BioEase tag and residual enterokinase. BioEase-tagged CTLD14 (7 μg / ml) was dispensed into microplate wells in 50 μl aliquots and coated overnight at 4°C. The CTLD14 solution was discarded, and the wells were washed three times with 200 μl of PBS. 250 μl of blocking buffer (0.25% BSA / PBS) was added and blocked for 2 hours at 25°C. The wells were then washed three times with 200 μl of PBS. Next, a 0.0075% BSA / PBS solution containing fully oxidized LDL (f-OxLDL) at 0–250 ng / ml was prepared as a standard for plotting a calibration curve, and 100 μl of this solution was added to the standard wells. To the same plate, 100 μl of the subject serum or plasma diluted 1:1000 with PBS was added to each well. After 2 hours of incubation at 25°C, the wells were washed five times with 200 μl of PBS, and 100 μl of anti-LDL chicken polyclonal antibody (diluted 1:6000 in blocking buffer) was added per well. After 1 hour of incubation at 25°C, the wells were washed five times with 200 μl of PBS, and 100 μl of anti-chicken IgY-HRP conjugate (diluted 1:6000 in blocking buffer) was added per well. After 1 hour of incubation at 25°C, the wells were washed five times with 200 μl of PBS, and 50 μl of TMB solution (as HRP substrate) was added per well to confirm color development. The reaction was stopped by adding 50 μl of 1N HCl per well, and the absorbance at 450 nm was measured using a microplate reader.

[0131] (result) The results are shown in Figure 3. A method based on a sandwich ELISA using middle silk gland (MSG)-derived CTLD14 and chicken anti-polyclonal antibodies can quantify oxidized LDL in samples. When measuring actual human serum, it was confirmed that the amount of oxidized LDL in samples can be measured without being affected by abnormal values ​​such as higher than normal LDL, lower than normal HDL, or higher than normal TG, even when measuring samples (serum) that showed abnormal values ​​such as higher than normal LDL, lower than normal HDL, or higher than normal TG. However, this method requires overnight immobilization of CTLD14 and approximately 8 hours for subsequent measurement.

[0132] Example 4: Lateral Flow (Immunochromatography) Assay (material and method) (Preparation of metal colloid-labeled CTLD14) Platinum colloid (wine red chemical, OD = 12) was transferred to a silicone-coated tube, and an equal volume of 10 mM Tris (pH 8.5) was added and ultrasonically dispersed. CTLD14 (30 μg / ml), which had been dialyzed against 5 mM Tris, pH 8.5, was added to the colloid solution, immediately vortexed, and then allowed to stand at room temperature for 15 min. An equal volume of conjugate buffer (1% BSA / 5 mM Tris, pH 8.5) was added to the tube, immediately vortexed, and then allowed to stand at room temperature for 15 min. The reaction mixture was centrifuged (9200 × g, 2 min), the supernatant was removed, and the conjugate buffer was added and the mixture was resuspended by ultrasonication. After allowing to stand at room temperature for 15 min, the mixture was centrifuged again, the supernatant was removed, and the mixture was resuspended in conjugate buffer containing 5% trehalose by ultrasonication. The mixture was then stored at 4°C. An outline of the preparation is shown in Figure 4.

[0133] (Preparation of immunochromatographic strips) On a half-strip membrane consisting of only an absorbent pad and membrane, 0.5 μl of streptavidin was spotted as a control spot, and 0.5 μl of single-chain antibody, chicken anti-LDL polyclonal antibody, or anti-ApoB antibody was spotted as a test spot, and then dried at 37°C for 1 hour. For blocking, 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, followed by another 5 minutes of gentle shaking. After washing, the strips were gently patted dry and air-dried overnight on a paper towel covered with Kimwipes. An overview of the preparation is shown in Figure 4.

[0134] (Detection of oxidized LDL by immunochromatographic assay) Oxidized LDL solution (0-3 μg) was added to 55 μl of 0.5% BSA / PBS solution and mixed with 10 μl of platinum colloid-modified CTLD (14). The sample was transferred to a microplate well, and the prepared immunochromatography half-strip was inserted. After 20 to 30 minutes, color development in the test and control spots was confirmed. Single-chain antibody (Figure 7), chicken polyclonal antibody (Figure 8), and anti-ApoB monoclonal antibody (Figure 9) were used in the test spots.

[0135] (result) As shown in Example 3, oxidized LDL can be detected (quantified) using ELISA-like techniques, but the measurement is time-consuming and cumbersome. The use of biotinylated CTLD14 labeled with metal colloids offers the potential for rapid detection of a wide range of oxidatively modified LDL, a risk factor for disease. Furthermore, the biotinylated CTLD14 has the advantage of allowing the use of streptavidin as a control line. However, the metal colloid modification process requires a slightly alkaline pH (pH 8.5–9.2), and conventional CTLD14 does not maintain its activity, making it difficult to prepare an effective detection reagent. Biotinylated CTLD14 derived from silkworm silk glands showed unexpected stability at alkaline pH, enabling the preparation of metal colloid-labeled CTLD14. Because MSG-derived CTLD14 could be modified with metal colloids without losing its activity under alkaline conditions, we performed circular dichroism (CD) spectroscopy of CTLD14 at alkaline pH. As a result, it was confirmed that MSG-derived CTLD14 maintained its α-helical structure at pH 7, 8, and 9. Contrary to expectations, MSG-derived CTLD14 maintained its structure even in a weakly alkaline buffer solution, confirming that modification with metal colloids is possible (Figure 6).

[0136] As shown in Figure 7, it was confirmed that the use of platinum colloid enabled better detection of oxidized LDL than the commonly used gold colloid. In Figure 7, a single-chain antibody with a BioEaseTag was used for the test line. Although single-chain antibodies are difficult to store for long periods (stable for about three weeks), the single-chain antibody with a BioEaseTag was unexpectedly stable.

[0137] When anti-LDL chicken polyclonal antibody was used as the test line, oxidized LDL could be detected only when CTLD14 was modified with platinum colloid, whereas no clear spot was observed with palladium colloid (Figure 8). The sensitivity required for detecting oxidized LDL in human serum is 2-3 μg / ml, but in cases of dyslipidemia, serum concentrations of 50 μg / ml or higher are expected. When converted to a lateral flow assay, detection of 30 ng / assay (90 μl solution) is possible, confirming that it is possible to detect elevated oxidized LDL concentrations even in actual human samples.

[0138] When palladium colloid was used, oxidized LDL could be detected by using anti-ApoB monoclonal antibody as the test line (Figure 9).

[0139] (Example 5: Detection of AGEs by immunochromatographic assay) (material and method) (Preparation of metal colloid-labeled sRAGE) Gold or palladium colloids (both wine red chemicals, OD = 12) were transferred to a siliconized tube, and the pH was adjusted with 10 mM Tris (pH 8.5 or 9.2) and then ultrasonically dispersed. sRAGE (60–90 μg / ml) dialyzed against 5 mM Tris, pH 8.5 (or pH 9.2) was added to the colloid solution, immediately vortexed, and then allowed to stand at room temperature for 15 min. Conjugate buffer (5 mM Tris, pH 8.5 or pH 9.2 containing 0.25–1.0% BSA, 0.05–1.0% PVA, or 0.05–2.5% casein) was added to the reaction mixture, immediately vortexed, and then allowed to stand at room temperature for 15 min. The reaction mixture was centrifuged (8000 × g for 2 min for gold colloids, 6000 × g for 2 min for palladium colloids) and the supernatant was removed. The conjugate buffer was added and the mixture was resuspended by ultrasonication. After leaving the mixture at room temperature for 15 minutes, it was centrifuged again to remove the supernatant, and the mixture was resuspended in conjugate buffer containing 5% trehalose by ultrasonication and stored at 4°C. A partially saponified aqueous solution of PVA (polyvinyl alcohol) was prepared from powder with a degree of polymerization of 500-2000 and a degree of saponification of 78.5-98.5.

[0140] (Preparation of immunochromatographic strips) On a half-strip membrane consisting of only an absorbent pad and membrane, 0.5 μl of streptavidin was spotted as a control spot, and 0.5 μl of anti-BSA or anti-OVA antibody was spotted as a test spot. The strip was then dried at 37°C for 30 min to 2 h. For blocking, the strip was immersed in blocking buffer (5 mM Tris or PBS solution containing 0.25-1.0% BSA, 0.05-1.0% / PBS PVA, or 0.05-2.5% casein) at room temperature for 15 min with gentle shaking, then gently washed twice with MilliQ water, followed by another 5 min with gentle shaking. After washing, the strip was gently patted dry and air-dried overnight on a paper towel covered with Kimwipes. An overview of the prepared strips and a schematic diagram of AGE detection are shown in Figure 10.

[0141] (Detection of AGEs by immunochromatographic assay) The prepared half-strips were confirmed to be capable of detecting AGEs. Specifically, 0.1 μg of AGE solution (Glucose-AGE-BSA) was added to 60 μl of reaction buffer (PBS containing 0.05–2.5% casein) and mixed with colloidal metal sRAGE. The resulting mixture was transferred to a microplate well, and the prepared immunochromatography half-strips (control spots coated with streptavidin, and test spots coated with four anti-BSA antibody concentrations) were inserted. After 20–30 minutes, color development was observed in the test and control spots. When whole blood was used as the sample, 5–50 μl of blood diluted 5–500 times with buffer was used. The sample preparation for whole blood use is shown in Figure 11.

[0142] (result) As shown in Figure 12, when palladium or gold colloid-modified sRAGE was used, color development of the control line was confirmed, confirming that development was normal. Furthermore, a test line spot was also confirmed, demonstrating that irritating AGEs can be detected. It was revealed that 1 μg of irritating AGEs can be detected, and since the concentration of irritating AGEs in the serum of healthy individuals is 5 to 10 μg / ml, it was confirmed that the present invention makes it possible to distinguish between healthy individuals and individuals with inflammatory bowel disease.

[0143] (Example 6: Possibility of detecting oxidized LDL in whole blood) (material and method) Whole blood and colloidal metal-labeled CTLD14 were added to a microtube containing a heparin sheet and PBS, mixed, and then transferred to a well in a microplate. A full strip (with streptavidin applied to the control spot) with a blood cell removal filter (LF1, FUSION5, MF1, or VF2) was inserted and allowed to spread vertically.

[0144] (result) The results are shown in Figure 11. Color development was confirmed in the control spots after development, confirming that the fabricated full strip can be used to develop samples even in whole blood. LF1, FUSION5, MF1, and VF2 were used as blood cell removal filters (from left to right), and development was possible with all of the filters without being affected by blood cells.

[0145] (Example 7: Possibility of detecting irritating AGEs in whole blood) (material and method) Whole blood and metal colloid-labeled sRAGE were added to a microtube containing a heparin sheet and PBS, mixed, and then transferred to a well of a microplate. A full strip (with streptavidin applied to the control spot) with a blood cell removal filter (LF1) attached was inserted and allowed to spread vertically.

[0146] (result) The results are shown in Figure 13. Palladium colloid-modified sRAGE was used as the detection agent for R3, R10, and R14, and gold colloid-modified sRAGE was used as the detection agent for R5 and R11. The blocking buffer used was 0.5% BSA / PBS for R3 and R5, 0.5% casein / PBS for R10 and R11, and 0.5% PVA / PBS for R14. Control spots were observed for R3, 5, 10, and 11, suggesting that membranes blocked with 0.5% BSA or 0.5% casein and palladium colloid-modified sRAGE or gold colloid-modified sRAGE as the detection agent could be developed without being affected by blood cells, potentially enabling the detection of irritant AGEs in whole blood. When PVA was used as the blocking agent, no control spots were obtained, as shown in R14, and development was unsuccessful.

[0147] Example 8: Detection of AGEs by lateral flow detection method using metal nanoparticles Preparation of metal nanoparticle-labeled sRAGE Gold colloid or palladium colloid (both from Morinaga Institute of Biological Sciences, OD = 12) was transferred to a siliconized tube, the pH was adjusted with 10 mM Tris (pH 8.5 or 9.2), and the mixture was sonicated. sRAGE (60-90 μg / ml) dialyzed against 5 mM Tris, pH 8.5 (or pH 9.2) was added to the colloid solution, vortexed immediately, and then allowed to stand at room temperature for 15 min. Conjugate buffer (5 mM Tris, pH 8.5 or pH 9.2 containing 1.0% BSA, 0.1-4.0% PVA, or 1-2.5% casein) was added to the reaction mixture, vortexed immediately, and then allowed to stand at room temperature for 15 min. The reaction mixture was centrifuged (8000 × g for 2 min for gold colloid, 6000 × g for 2 min for palladium colloid) and the supernatant was removed. The mixture was then resuspended in conjugate buffer and sonicated. After standing at room temperature for 15 minutes, the mixture was centrifuged again to remove the supernatant, and the mixture was resuspended in conjugate buffer containing 5% trehalose by ultrasonication and stored at 4°C.

[0148] The polyvinyl alcohol (PVA) powder used had a degree of polymerization of 500-2000 and a degree of saponification of 78.5-98.5, and was prepared as a partially saponified aqueous solution.

[0149] Lateral flow test strip preparation method 0.5 μl of streptavidin (Ctl spots) and 0.5 μl of AGE (e.g., glucose-AGE-BSA) were spotted on the appropriate areas of 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 the absorbent pad and membrane. The spots were then dried at 37°C for 30 min to 2 h. For blocking, the spots were immersed in blocking buffer (0.5-1.0% BSA, 0.05-2.0% / PBS PVA, or 0.1-1.0% casein in 5 mM Tris or PBS) at room temperature for 15 min with gentle shaking, washed twice with MilliQ water, and then gently shaken for another 5 min. After washing, the strips were gently patted dry and air-dried overnight on a paper towel covered with Kimwipes.

[0150] Detection of AGEs by lateral flow detection method 0.2-4 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 (PBS containing 0.5% BSA, 0.05-2.0% / PBS PVA, or 0.1-1% casein, or 5 mM Tris, pH 8.5, or 5 mM Tris, pH 9.2) and mixed with colloidal metal sRAGE. The resulting mixture was transferred to a microplate well, and a prepared lateral flow half strip was inserted. After 20 to 30 minutes, color development in the test and control spots was confirmed. When using whole blood as a sample, 75 μl of blood diluted 500x with reaction buffer was added to the AGE solution and mixed with the colloidal conjugate. The mixed solution 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 had passed and the strip had completely absorbed the mixture, 50 μl of reaction buffer was added to the wells, and after another 30 minutes or so, the color development in the test and control spots was confirmed.

[0151] Detection of AGEs in whole blood Fructose-treated BSA (antagonistic AGEs) was applied to a test spot, and a sample (glycolaldehyde-treated BSA) was added to fresh human blood and developed. Binding of metal colloid-labeled biotinylated sRAGE to the test line was inhibited by AGEs in whole blood, and a decrease in color development was confirmed visually (Figure 14A). Glucose-treated BSA (antagonistic AGEs) was applied to a test spot, and a sample (glycolaldehyde-treated BSA) was added to fresh human blood and developed. Binding of metal colloid-labeled biotinylated sRAGE to the test line was inhibited by AGEs in whole blood, and a concentration-dependent decrease in color development was confirmed visually (Figure 14B). An overview of the detection system in which antagonistic AGEs were applied to the test spot is shown in Figure 15.

[0152] (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. 2019-203441 filed on November 8, 2019, and Japanese Patent Application No. 2019-224098 filed on December 11, 2019, and the contents thereof are incorporated by reference into this specification. [Industrial Applicability]

[0153] The present disclosure is useful in the field of disease diagnosis or predictive diagnosis industry. [Sequence List Free Text]

[0154] 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

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 a metal colloid as part of a membrane or as a separate element, wherein the biomarker molecule is AGEs, the abnormal form of the biomarker molecule is 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 of 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 according to any one of claims 1 to 3, wherein the metal colloid is provided as a detection reagent.

5. The device or kit according to any one of claims 1 to 4, wherein the metal colloid is 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 metal colloid is selected from the group consisting of gold, silver, platinum and palladium.

9. The device or kit of claim 8 , wherein the metal colloid is a platinum colloid.

10. The device or kit according to any one of claims 1 to 9, wherein the sample is a blood sample.

11. The device or kit of claim 10, wherein the blood sample is serum or whole blood.

12. 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 metal colloid, the binding molecule being capable of forming a conjugate with an aberrant form of a biomarker molecule or a competitor molecule thereof; A step of contacting the mixed sample with the sample contact portion of the device or kit according to any one of claims 1 to 11; After contacting, adding a buffer solution; A method comprising:

13. 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 metal colloid, 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: 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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