Lateral flow device for detecting acute aortic syndrome

Abstract

The present invention relates to a lateral flow device for detecting at least one protein (AAD protein) released during an event leading to acute aortic syndrome / acute aortic dissection (AAS / AAD) in a human / patient body fluid sample. The device comprises a strip, a sample pad for receiving a body fluid sample, a first conjugate pad having a conjugate containing a protein antibody that binds to the AAD protein, and a first detection band containing the first conjugate-binding protein, the presence of the AAD protein in the sample is indicated by a visible color change. Here, the AAD protein is selected from a group of AAS / AAD biomarkers, including D-dimer, smooth muscle actin myosin isozyme, CK-MB, and nucleic acid component (DISAMIN). Furthermore, the present invention also relates in other aspects to a method for detecting the AAD protein in a patient's blood using the lateral flow device according to the present invention, a kit comprising the device, and a method for determining the presence of AAD in a patient using an immunoassay.

Description

Technical Field

[0001] The present invention relates to a lateral flow device for detecting at least one protein (AAD-protein) released during events leading to acute aortic syndrome / acute aortic dissection (AAS / AAD) in a human / patient's body fluid sample. The device comprises a strip, a sample pad for receiving the body fluid sample, a first conjugate pad having a conjugate containing a protein antibody that binds to the AAD-protein, and a first detection band containing a first conjugate-binding protein. The presence of the AAD-protein in the sample is indicated by a visible color change. The AAD-protein is selected from the group of biomarkers of AAS / AAD including D-dimer, smooth muscle actin, myosin isozyme CK-MB, and nucleic acid component (DISAMIN). Further, in other aspects, the present invention also relates to a method for detecting an AAD-protein in a patient's blood using the lateral flow device according to the present invention, a kit containing the device, and a method for determining the presence of AAD in a patient using an immunoassay.

Background Art

[0002] Acute aortic dissection (AAD) is a life-threatening clinical condition requiring emergency surgical intervention and is an aspect of acute aortic syndrome (AAS). The incidence of aortic dissection is reported to peak in the 50s and 60s, with 2,000 new cases reported annually in North America and 3,000 in Europe (Non-Patent Literature 1, 2). The triggers for the onset are intimal lacerations due to congenital defects of the aortic wall such as Marfan syndrome (MFS), Ehlers-Danlos syndrome (a group of heterogeneous connective tissue disorders), idiopathic Erdheim-Güssel medial necrosis, and bicuspid aortic valve (the most common congenital heart defect), or acquired intimal injury due to hypertension, chest trauma, or other causes of unknown origin (Non-Patent Literature 3-5). Intimal lacerations occur due to subintimal or medial necrosis, and eventually blood enters the aortic wall, causing the layers of the aortic wall to separate and the aortic dissection to progress rapidly. Separation of the media and adventitia of the aortic wall can lead to perfusion failure of many organs and catastrophic complications (Non-Patent Literature 2, 6). An important clinical fact is that aortic dissection can occur in both aortic diameters dilated by aneurysms and normal diameters. The most feared complication of aortic dissection is outward rupture, which carries a high mortality rate (Non-Patent Literature 7). Surgical treatment varies depending on the location of the initial tear and the longitudinal extent of the dissection. Various classifications have been proposed to describe dissections, but the DeBakey and Stanford classifications are more commonly used (Non-Patent Literature 8, 9). When the ascending aorta is affected (Stanford type A), the mortality rate in untreated patients is approximately 36-72% within 48 hours of onset and approximately 62-91% within one week of onset (Non-Patent Literature 10-13). Symptoms of ADD, such as chest pain, are often confused with those of myocardial infarction, complicating diagnosis and delaying treatment of AAD. Therefore, a deeper understanding of the molecular mechanisms underlying AAD could be the first step in supporting the development of rapid tests for monitoring high-risk patients in the future.

[0003] Given this fact, providing a diagnostic antibody test kit that can adequately detect one or more proteins released during events causing acute aortic syndrome / acute aortic dissection in human / patient samples is extremely beneficial, especially since time is critical in this indication.

[0004] Therefore, providing a lateral flow test is particularly desirable. A lateral flow (LF) test is a membrane (strip)-based immunoassay test. A lateral flow device has a test strip made of a membrane, such as porous paper or sintered polymer, that allows for capillary flow of the sample and detection reagent. The membrane (strip) also holds material that forms the test line and control line. One end of the membrane (strip) is fitted with a sample pad that contacts another pad used to store the detection conjugate. The other end of the membrane (strip) has a wicking pad that holds the liquid and reagent that has moved through the membrane (strip) by capillary action. Lateral flow devices exist for a wide range of targets, including the detection of infectious agents, metabolites, antibodies, toxins, and drugs used in human and veterinary applications.

[0005] Lateral flow tests generally model themselves after existing immunoassays and can be performed as sandwich or competitive assays. While various assay variations are possible, a positive signal is typically obtained by the specific accumulation of the detection complex in the test line.

[0006] Lateral flow devices typically need to be specially developed through an iterative process to suit their intended application. Each component of a lateral flow device test strip must be individually evaluated and tested to determine the optimal combination of components. Identifying the optimal membrane is a crucial step in test strip development, as it determines the capillary flow characteristics that must be suitable for the type of sample being tested.

[0007] In this regard, a rapid, reliable, and accurate point-of-care lateral flow test is extremely useful, and the present invention provides precisely this. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Isselbacher EM. Disease of the aorta. Heart Disease, 6th edn. Braunwald E, Zipes DP, Libby P, Eds. WB Saunders Company, Philadelphia, 2001;1422-56. [Non-Patent Document 2] Nienaber CA, Fattori R, Mehta RH, Richartz BM, Evangelista A, Petzsch M, et al. The International Registry of Acute Aortic Dissection (IRAD). Gender-Related differences in acute aortic dissection. Circulation. 2003;109:3014-21. [Non-Patent Document 3] Pearson GD, Devereux R, Loeys B, Maslen C, Milewicz D, Pyeritz R, Ramirez F, et al. Report of the National Heart, Lung, and Blood Institute and National Marfan Foundation Working Group on research in Marfan syndrome and related disorders. Circulation. 2008;118: 785-91. [Non-Patent Document 4] Parapia LA, Jackson C. Ehlers-Danlos syndrome-a historical review. Br J Haematol. 2008;141:32-5.

Non-licensed Document 5

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Non-licensed Document 7

Non-licensed literature 9

[0009] As mentioned above, intimal tears due to defects in the aortic wall trigger the onset of aortic dissection. Dissection of these layers occurs after aortic rupture. Five to ten minutes after the onset of pain, biological markers (e.g., DISAMIN: D-dimer, smooth muscle actin, myosin isozymes, CK-MB, nucleic acid components; HMGI-C, aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, etc.) are released into the bloodstream. Biomarker levels remain high for 6 to 10 days after the onset of pain, making them effective for the early detection of aortic wall injury. Biomarker measurement has been validated during surgical intervention to show high specificity for aortic wall injury, demonstrating its usefulness as a biomarker suggesting aortic dissection.

[0010] Furthermore, identifying proteins in the blood (including serum / plasma) that differ in elevation / concentration between patients with aortic dissection and patients with myocardial infarction through these tests could bring significant benefits. It should be noted that since myocardial infarction (MI) and acute myocardial infarction (AAD) often present with similar symptoms, the gold standard for diagnosing myocardial infarction (MI) to date has been based on "STE" in 12-lead electrocardiogram diagnosis. [Means for solving the problem]

[0011] In one embodiment, the present invention provides a lateral flow device for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian body fluid sample. The device comprises the following components: i) A strip formed with a material that allows capillary flow along a portion of the strip; ii) A sample pad positioned near one end of the strip for receiving a bodily fluid sample; iii) A first conjugate pad positioned within the strip, wherein during operation, the sample flows through the strip by capillary action from the sample pad to the conjugate pad, mobilizing the conjugate contained in the conjugate pad, the conjugate containing a protein antibody conjugated to the detection agent and bound to the 1-AAD-protein, and iv) A first detection band comprising a first conjugate-binding protein fixed within the strip along a band positioned substantially perpendicular to the direction of sample flow within the strip, wherein when a recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the first conjugate-binding protein in the first detection band, the presence of the 1-AAD-protein in the sample is indicated by a visible color change, Here, the 1-AAD-protein is selected from a group of AAD biomarkers including D-dimer, smooth muscle actin myosin isozyme, CK-MB, and nucleic acid component (DISAMIN), and is particularly selected from HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin. In preferred embodiments, the 1-AAD-protein is aggrecan, HMGI-C, or DISAMIN (D-dimer, smooth muscle actin myosin isozyme CK-MB, and nucleic acid component), and in very preferred embodiments, the 1-AAD-protein is HMGC-I. The lateral flow device further comprises at least one second protein antibody that binds to a second protein (2-AAD-protein) potentially released during events leading to acute aortic dissection. Preferably, the 2-AAD-protein is selected from a group of AAD biomarkers including D-dimer, smooth muscle actin myosin isozyme CK-MB, and nucleic acid component (DISAMIN). In particular, the 1-AAD-protein is selected from aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, or D-dimer, and is especially preferably D-dimer. Therefore, the 1-AAD-protein is HMGC-I, the 2-AAD-protein is D-dimer, and it is also preferable that the protein antibody that binds to the 1-AAD-protein binds to the antigen of the amino acid sequence or fragment of SEQ ID NO: 1. As described above, in the most preferred embodiment, the 1-AAD-protein is HMGI-C, and the 2-AAD-protein is D-dimer. This is a very preferred embodiment of the lateral flow device of the present invention, comprising at least a first antibody against HMGI-C (1-AAD-protein) and a second antibody that binds to the D-dimer (2-AAD-protein). This combination makes it possible to distinguish between AAD and MI very well, and the combination of HMGI-C and D-dimer greatly improves the detection accuracy of AAD.

[0012] In a further embodiment, the lateral flow device according to the invention further comprises another MI detection band comprising at least one MI-protein antibody that binds to a protein that is an indicator of acute myocardial infarction (MI-protein), and a further conjugate-binding protein different from the first and second immobilized conjugate-binding proteins immobilized within the strip, the band being located substantially perpendicular to the direction of flow of the sample along the strip. Thereby, when at least one immobilized MI-protein antibody sample conjugate in the sample contacts the further conjugate-binding protein in the MI-detection band, the presence of the MI-protein in the sample is indicated by a visible color change. Preferably, the MI-protein is selected from the group consisting of creatine phosphokinase (CK) or its MB isozyme, cardiac myosin light chain, troponin T (TnT), and human heart fatty acid binding protein. This is very important. Because MI and AAD often exhibit the same symptoms, the gold standard for MI diagnosis to date has been the elevation of "STE" in the electrocardiogram. In this embodiment, the MI-protein (to which the MI-protein antibody binds) is very preferably troponin T (TnT) or troponin I (ThI), or troponin T (TnT). Because MI and AAD often exhibit the same symptoms, this enables good discrimination between AAD and MI.

[0013] Also, in this embodiment, the MI-protein (to which the MI-protein antibody binds) is troponin T (TnT) or troponin I (ThI), or troponin T (TnT), and it is very preferable that other MI-proteins are creatine phosphokinase (CK), particularly its MB isozyme (CK-MB). This combination enables very good identification of MI, and the detection accuracy of myocardial infarction is improved (e.g., 91% or more) by the combination of troponin and CK-MB. Also, because MI and AAD often exhibit the same symptoms, good discrimination between AAD and MI is also possible.

[0014] Most preferably, in this embodiment, the lateral flow device according to this embodiment is defined as follows: - The lateral flow device further comprises an MI-protein antibody that binds to an MI-protein which is troponin T-like protein (TnT) or troponin I (TnI), or troponin T (TnT); - The 1-AAD-protein is HMGI-C; and - The lateral flow device further comprises another protein antibody that binds to another protein potentially released during the event leading to acute aortic dissection (X-AAD-protein or 2-AAD-protein). Here, the X-AAD-protein (or 2-AAD-protein) is a D-dimer.

[0015] Therefore, the lateral flow device is composed of a primary antibody against HMGI-C (1-AAD-protein), a secondary antibody that binds to troponin (MI-protein), and a tertiary antibody that binds to D-dimer (X-AAD-protein or 2-AAD-protein). This combination makes it possible to very well distinguish AAD from MI, and the combination of HMGI-C and D-dimer greatly improves the detection accuracy of AAD.

[0016] Most preferably, in this embodiment, the lateral flow device according to this embodiment is defined by the following: - The lateral flow device further comprises two MI-protein antibodies, one antibody binds to MI-protein troponin T (TnT) or troponin I (TnI) (especially troponin T (TnT)), and the other antibody binds to MI-protein creatine phosphokinase (CK), especially its MB isozyme (CK-MB); - The 1-AAD-protein is HMGI-C; and - The lateral flow device further includes another protein antibody that binds to another protein that may be released during the event leading to acute aortic dissection (X-AAD-protein or 2-AAD-protein), where X-AAD-protein or 2-AAD-protein is a D-dimer.

[0017] Therefore, the lateral flow device of the present invention comprises a first antibody against HMGI-C (1-AAD-protein), a second antibody that binds to troponin (MI-protein), a third antibody that binds to D-dimer (X-AAD-protein or 2AAD-protein), and a fourth antibody that binds to CK, particularly its MB isozyme (CK-MB). This combination enables very good differentiation between AAD and MI, the combination of HMGI-C and D-dimer significantly improves the detection accuracy of AAD, and the combination of troponin and CK-MB improves the detection accuracy of myocardial infarction (e.g., over 91%).

[0018] In a further embodiment, the lateral flow device according to the present invention further comprises a control line positioned substantially perpendicular to the direction of sample flow along the strip.

[0019] In another further embodiment, a lateral flow device according to any of the embodiments described above, wherein acute aortic dissection (AAD) includes aortic dissections including acute type A aortic dissection, and / or one or more of the following aortic dissections / acute aortic syndromes: thoracic aortic aneurysm, chronic dissection, branched and fenest type stent graft indications, such as penetrating aortic ulcer and intradural hematoma; various treatments for aortic arch dissection complicated with Kommerell diverticulum; secondary intervention after endovascular repair of aortic dissection; pseudoaneurysm complicated with internal carotid artery dissection; establishment of indications for invasive treatment for thoracic aortic aneurysm; aortic arch dissection: classification controversy; treatment algorithms for patients with (sub)acute type B aortic dissection; descending thoracic aortic dissection; differences in aortic disease between patients with bicuspid aortic valve and patients without aortic aneurysm; angina chest pain; dyspnea with hypertensive crisis; sudden coma or shock of unknown cause.

[0020] Another aspect of the present invention is a kit for detecting the protein (AAD protein) released during an event leading to acute aortic dissection (AAD protein) in a mammalian body fluid sample, the kit comprising: a) Lateral flow assay devices as described above and below; and b) Instructions for using the lateral flow assay devices described above and below.

[0021] Further embodiments of the present invention also include a method for detecting the AAD-protein in mammalian body fluids, which involves using the lateral flow assay devices described above and below.

[0022] A further aspect of the present invention includes a method for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian body fluid sample. This method includes the following steps: a) The step of placing the sample on the sample pad of the lateral flow assay device. The lateral flow assay device includes the following: (1) A strip formed of a material that allows capillary flow along a portion of the strip; (2) A sample pad positioned near one end of the strip for receiving a bodily fluid sample; (3) A first conjugate pad positioned within the strip, wherein during operation, the sample flows through the strip by capillary action from the sample pad to the conjugate pad, mobilizing the conjugate contained in the conjugate pad, the conjugate containing a protein antibody conjugated to the detection agent and bound to the 1-AAD-protein, (4) A first detection band comprising a first conjugate-binding protein fixed along a band positioned substantially perpendicular to the direction of sample flow along the strip, wherein when a recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the first conjugate-binding protein in the first detection band, the presence of the 1-AAD-protein in the sample is indicated by a visible color change. Here, the 1-AAD-protein is selected from a group of AAD biomarkers including D-dimer, smooth muscle actin myosin isozyme, CK-MB, and nucleic acid component (DISAMIN), and is particularly selected from HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, preferably HMGI-C, and the antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1. b) The presence of labeled particles in the detection region is examined to detect whether the AAD-protein is present in the sample.

[0023] The present invention also includes a method for determining acute aortic dissection (AAD) in a patient by immunological measurement, the method comprising: a) Take a sample of the patient's bodily fluids. a. At least one antibody against the first protein (1-AAD-protein) released during the event leading to acute aortic dissection, and b. Incubate with binding partner B for either the 1-AAD- protein or the antibody, Here, either the antibody or the binding partner B is bound to the detection agent. It forms an immunological complex containing the detection agent; b) Isolate the immunological complex from the remaining sample; and c) Determine the presence of the detection agent in the isolated complex or in the remaining sample as an indicator of the 1-AAD- protein in the sample; This determines the occurrence of AAD in the patient. Here, the 1-AAD-protein is selected from a group of AAD biomarkers including the D-dimer smooth muscle actin myosin isozyme CK-MB and nucleic acid components (DISAMIN), particularly HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, preferably HMGI-C, and the antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 is a schematic diagram of a lateral flow device according to the present invention. The device (1) has a sample pad (2), a conjugate pad (3) containing a mobile conjugate containing an antibody protein conjugated to an AAD-protein conjugated to a detection agent, a membrane (4), and an optional wicking pad (7) for receiving and holding fluid that has passed from the sample pad (2) through the conjugate pad (3) and membrane (4) by capillary flow. A detection band (5) containing an immobilized binding protein is shown. Band (6) is an optional positive control band. The device (1) may further include a backing (8). [Figure 2] Figure 2 is a schematic diagram of various methods for injecting a sample into the lateral flow device according to the present invention. a) For plasma and serum samples. b) For venous whole blood. c) For whole blood collected by finger puncture: capillary method. d) For whole blood collected by finger puncture: hanging drop method. [Figure 3]Figure 3 shows a schematic example of the AAD-protein, the conjugate from the conjugate pad, and the first conjugate-binding protein complex bound to the reporter for the visible color change in the detection band. A: Magnetic beads B: Capture antibody (protein antibody that binds to AAD-protein) C: AAD-protein (biomarker) D: Biotinylated detection antibody (conjugate-binding protein) E: Streptavidin F: Phycoerythrin fluorescent reporter [Figure 4] Figure 4 shows the protein sequence of HMGI-C (high mobility protein), consisting of 147 amino acids, and sequence number 1. [Figure 5] Figure 5 shows the protein sequences of HMGI-C (high mobility group protein) with various tags used to express, isolate, and purify the protein HMGI-C in Example 1. [Figure 6] Figure 6 is a photograph of the gel showing the purity of protein HMGI-C obtained in Example 1. The conditions were reduced SDS-PAGE with Coomassie blue staining, and 2 μg of sample was loaded per lane. MW. Molecular weight marker. [Modes for carrying out the invention]

[0025] Other objects, aspects, features, and advantages of the present invention will become apparent from the following description. However, please understand that the detailed description and specific examples are for illustrative purposes only, as they illustrate preferred embodiments of the present invention, and various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0026] Detailed description of the invention definition Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” shall be understood to mean that the steps, elements, or groups of steps or elements described are included, but not that other steps, elements, or groups of steps or elements are excluded. Furthermore, terms containing “comprise,” “comprises,” and “comprising” may be limited to “consists of,” “consists of,” and “consisting of,” in which case they shall be understood to mean that the steps, elements, or groups of steps or elements described are included, but not that other steps, elements, or groups of steps or elements are excluded.

[0027] The terms “protein,” “protein fragment,” “polypeptide,” and “peptide” may be used interchangeably herein to refer to polymers of amino acid residues, as well as their variants, synthetic analogues, and natural analogues. Therefore, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-natural amino acids, such as chemical analogues of the corresponding natural amino acids, as well as to natural amino acid polymers and their natural chemical derivatives.

[0028] The term "antibody," also known as "immunoglobulin," refers to a Y-shaped protein of the immune system that specifically identifies foreign substances or antigens, such as components of bacteria, yeast, parasites, and viruses. Each end of the "Y" in an antibody contains an antigen-binding site specific to a particular epitope on the antigen, enabling these two structures to bind precisely. Antibodies typically contain all or part of an Fc region to facilitate detection by Fe-binding proteins, and may also contain one or more antigen-binding sites to facilitate detection by antibody-specific conjugates such as antigens or antigen peptides. Antibodies can be of types such as IgG, IgE, IgD, IgM, or IgA.

[0029] An "antigen" refers to a molecule with distinct surface characteristics or an epitope that can stimulate a specific immune response. Antibodies (immunoglobulins) are produced by the immune system in response to exposure to an antigen. Antigens include proteins, carbohydrates, and lipids, but since carbohydrates and lipids cannot induce an immune response on their own, only protein antigens are classified as immunogens.

[0030] The "antigen-binding site" or "binding region" of an antibody refers to the part of the immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable region ("V") of the heavy chain ("H") and light chain ("L"). Three distinctly different regions within the V region of the heavy and light chains are called "high-frequency variable regions," and these are flanked by more conserved adjacent regions called "framework regions" (FR). In an antibody molecule, the three high-frequency variable regions of the light chain and the three high-frequency variable regions of the heavy chain are arranged relative to each other in three-dimensional space, forming the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three high-frequency variable regions of the heavy and light chains are called "complementarity-determining regions" (CDR).

[0031] The term "Fc-binding protein" refers to a protein or polypeptide that can bind to the fragment crystallization region (Fc region) of an antibody. The Fc region is the terminal region of an antibody that interacts with cell surface Fc receptors and specific proteins of the complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the antibody's two heavy chains. The Fc regions of IgG and IgE contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc region of IgG antibodies has a highly conserved N-glycosylation site. The N-glycan bound to this site is primarily a core-fucosylated complex-type branched structure. Furthermore, these N-glycans also contain small amounts of bisecting GlcNAc and α-2,6-linked sheaphosphate residues. The Fab region of an antibody contains a variable section that defines the antibody's target specificity, in contrast to the Fc region of all antibodies in a class, which is identical in each species and is constant rather than variable.

[0032] "Nanoparticles" refer to uniform particles with a size of 1 to 200 nm.

[0033] The term "nanoshell" refers to nanoparticles consisting of a core and a metal shell (usually gold).

[0034] In the context of this invention, the term “acute aortic dissection (AAD)” is understood to refer to a clinical entity that is part of “AAS,” and is caused by intimal laceration due to congenital defects of the aortic wall, such as Marfan syndrome (MFS), Ehlers-Danlos syndrome (a group of heterogeneous connective tissue disorders), idiopathic Erdheim-Güssel medial necrosis, bicuspid aortic valve (the most common congenital cardiac dissection), or acquired intimal injury caused by hypertension, chest trauma, or other unknown mechanisms (Non-Patent Literature 3-5). One complication of aortic dissection is outward rupture, which is treated surgically. In the case of Stanford type A, the mortality rate for untreated patients is approximately 36%–72% within the first 48 hours and 62%–91% within the first week (Non-Patent Literature 10-13).

[0035] In the context of this invention, the term “acute aortic syndrome (AAS)” is understood as a broader term that includes AAD. AAS (and AAD) includes the following syndromes: perforated aortic ulcer and intramural hematoma, thoracic aortic aneurysm, chronic dissection – indications for treatment with branched and windowed stent grafts, various treatments for aortic arch dissection with Comerrell diverticulum, secondary interventions after endovascular repair of aortic dissection, pseudoaneurysm complicated with internal carotid artery dissection, established indications for invasive treatment of thoracic aortic aneurysm, aortic arch dissection: classification controversy, treatment algorithms for patients with (sub)acute type B aortic dissection, descending thoracic aortic dissection, differences in aortic disease in patients with bicuspid valves, aortic valve with or without aortic stenosis, angina pectoris, chest pain, dyspnea with hypertensive attacks, sudden coma or shock of unknown cause.

[0036] In the context of this invention, the term "1-AAD-protein" refers to one or more proteins released into the bloodstream after the onset of acute aortic syndrome / acute aortic dissection (AAS / AAD). This onset is an intimal tear due to a defect in the aortic wall. Dissection of these layers occurs after aortic rupture. One or more of these biological markers, "1-AAD-protein," are released into the bloodstream 5 to 10 minutes after the onset of pain. The concentration of the biomarker ("1-AAD-protein") remains high for 6 to 10 days after the onset of pain, providing an excellent one-time detection period for detecting aortic wall injury. The high specificity of biomarker measurement for aortic wall injury has been validated during surgical intervention, resulting in a biomarker that suggests aortic dissection. "1-AAD-protein" can be, for example, DISAMIN (D-dimer, smooth muscle actin, myosin isozyme, CK-MB, nucleic acid component), but may also be HMGI-C, aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, etc. HMGI-C is preferred.

[0037] In the context of this invention, the term “2-AAD-protein” refers to one or more proteins released into the bloodstream after the onset of acute aortic syndrome / acute aortic dissection (AAS / AAD). This onset is an intimal tear due to a defect in the aortic wall. Dissection of these layers occurs after aortic rupture. One or more of these biological markers, “2-AAD-protein,” are released into the bloodstream 5–10 minutes after the onset of pain. Since biomarker (“2-AAD-protein”) levels remain elevated for 6–10 days after the onset of pain, this provides an excellent one-time detection period for detecting aortic wall injury. The high specificity of biomarker measurement for aortic wall injury has been validated during surgical intervention, and as a result, it serves as a biomarker suggestive of aortic dissection. "2-AAD-protein" is, for example, DISAMIN (D-dimer smooth muscle actin myosin isozyme CK-MB and nucleic acid components), but it could also be HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin.

[0038] In the context of this invention, the term "X-AAD-protein" refers to one or more proteins released into the bloodstream after the onset of acute aortic syndrome / acute aortic dissection (AAS / AAD). This onset is an intimal tear due to a defect in the aortic wall. Dissection of these layers occurs after aortic rupture. One or more of these biological markers, "X-AAD-protein," are released into the bloodstream 5 to 10 minutes after the onset of pain. The concentration of the biomarker ("X-AAD-protein") remains high for 6 to 10 days after the onset of pain, providing an excellent one-time detection period for detecting aortic wall injury. The high specificity of biomarker measurement for aortic wall injury has been validated during surgical intervention, resulting in a biomarker that suggests aortic dissection. The "X-AAD-protein" is, for example, DISAMIN (D-dimer smooth muscle actin myosin isozyme CK-MB and nucleic acid components), but it could also be HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin.

[0039] In the context of the present invention, the term “biomarker” refers to any biological compound whose detection suggests the occurrence of an event in a living organism. In the present invention, “biomarker” is understood to mean, in particular, one or more proteins, especially enzymes, present in a biological sample of acute aortic dissection (AAD), and more specifically, the aforementioned “1-AAD-protein,” “2-AAD-protein,” or “X-AAD-protein.”

[0040] In the context of this invention, the term “sample pad” is understood as an area on (or part of) a strip onto which a liquid sample or analyte, preferably a bodily fluid sample, particularly a blood sample, such as human / patient blood, is applied. This sample pad may consist of a polymer such as sintered polymer, cotton, porous paper, microstructured polymer, or nitrocellulose, paper, or cotton. The sample pad should be configured to allow application of the bodily fluid, for example, through an opening in a lateral flow device. The sample pad may also function as a sponge and can often hold excess liquid.

[0041] In the context of this invention, the term “body fluid” is understood to mean all bodily fluids, particularly all bodily fluids in mammals such as humans and patients. Body fluids include blood, urine, semen, sputum, saliva, etc., but most preferably blood such as whole blood or serum.

[0042] In the context of the present invention, the term “strip” is understood as a material or combination of materials that enables capillary flow of a fluid along at least a portion thereof. The strip may have, and preferably has, the ability to spontaneously transport fluids such as bodily fluids or blood. The strip may include, or be composed of, polymers or papers such as sintered polymers, microstructured polymers, nitrocellulose, porous paper, and particularly porous paper such as nitrocellulose.

[0043] In the context of the present invention, the term “conjugate” is understood to mean a protein antibody conjugated to a biomarker (see above) with a detection agent. In preferred embodiments, the protein antibody is an antibody conjugated to 1-AAD-protein, 2-AAD-protein, or X-AAD-protein (see above) and conjugated to a detection agent (tag) such as gold or colored latex. Often, the antibody is lyophilized and / or a monoclonal antibody. Preferably, the antibody in the conjugate is an antibody conjugated to HMGI-C, more preferably a monoclonal antibody conjugated to HMGI-C, and most preferably a lyophilized monoclonal antibody conjugated to HMGI-C. Preferably, in the conjugate, this antibody is conjugated to gold particles or colored latex particles, more preferably to gold particles. Most preferably, the conjugate is a monoclonal antibody conjugated to gold particles and conjugated to HMGI-C.

[0044] In the context of the present invention, the term "conjugate pad" is understood as a region on (or part of) a strip through which a fluid sample or analyte, pre-coated on a sample pad, is transported, for example, by capillary action within the strip. The conjugate pad includes a conjugate (see above). This conjugate pad may be composed of a polymer or paper, such as a sintered polymer, porous paper, microstructured polymer, or nitrocellulose. The conjugate contained in the conjugate pad is mobilized as the sample or analyte flows through the strip from the sample pad to the conjugate pad by capillary action.

[0045] In the context of the present invention, the term “mobilize” means to mobilize from its current position, for example, within a conjugate pad. Preferably, this refers to the mobilization of a conjugate (see above) contained within a conjugate pad along the strip with the sample / fluid, for example, by capillary force toward / towards a “detection band”. Most preferably, this refers to the mobilization of a conjugate (see above) contained within a conjugate pad, bound to a biomarker (1-AAD-protein, 2-AAD-protein, or X-AAD-protein) from the sample / analyte via a protein antibody, along the strip with the sample / fluid.

[0046] In the context of the present invention, "1-AAD-protein antibody conjugate" (or "2-AAD-protein antibody conjugate") refers to a conjugate (see above) of a "protein antibody" that binds to the "biomarker" 1-AAD-protein (or 2-AAD-protein) and a "detection agent". This also includes a conjugate (see above) of a "protein antibody" that binds to the "biomarker" 1-AAD-protein (or 2-AAD-protein) and a "detection agent" that is bound to the biomarker 1-AAD-protein (or 2-AAD-protein) via that protein antibody.

[0047] In the context of the present invention, a "mobilized 1-AAD-protein antibody conjugate" (or a mobilized 2-AAD-protein antibody conjugate) is a conjugate (see above) of a "protein antibody" that binds to the "biomarker" 1-AAD-protein (or 2-AAD-protein) and a "detector," which is mobilized, for example, from a conjugate pad and moves with the sample. This also includes a conjugate (see above) of a "protein antibody" that binds to the "biomarker" 1-AAD-protein (or 2-AAD-protein) and a "detector." The detection agent binds to the biomarker 1-AAD-protein (or 2-AAD-protein) via its protein antibody and is mobilized, for example, from a conjugate pad and moves with the sample.

[0048] In the context of the present invention, the term "protein antibody" is understood to mean an antibody that is a protein that binds to a biomarker (see above). In preferred embodiments, the protein antibody is an antibody that binds to 1-AAD-protein, 2-AAD-protein, or X-AAD-protein (see above). Often, the antibody is lyophilized and / or a monoclonal antibody. Preferably, the protein antibody is an antibody that binds to HMGI-C, and more preferably a monoclonal antibody that binds to HMGI-C. Also, the protein antibody is an antibody that binds to a D-dimer, and more preferably a monoclonal antibody that binds to a D-dimer. Also, the protein antibody is an antibody that binds to the antigen or a fragment of the amino acid sequence of SEQ ID NO: 1, and more preferably a monoclonal antibody that binds to the antigen or a fragment of the amino acid sequence of SEQ ID NO: 1.

[0049] In the context of the present invention, the term "protein antibody binding to" means a protein antibody whose binding to an antigen is clearly defined. Typically, the "protein antibody binding to" binds to a biomarker (see above), and in preferred embodiments, the protein antibody binds to 1-AAD-protein, 2-AAD-protein, or X-AAD-protein (see above) as the antigen. Preferably, it is a protein antibody that binds to HMGI-C, and more preferably, it is a monoclonal antibody that binds to HMGI-C.

[0050] In the context of the present invention, the term “detector” is understood to mean gold, colored latex, or a tag such as gold and colored latex particles or magnetic beads. Preferably, the detector is part of the conjugate described above, preferably gold or latex particles conjugated to a protein antibody (see above).

[0051] In the context of this invention, the term “detection band” is understood as a region on a strip where a fluid sample or analyte contained in a conjugate pad is transported, for example, by capillary flow within the strip, along with the recruited conjugate (including “protein antibody”). The detection band is positioned substantially perpendicular to the direction of sample flow along the strip. The detection band contains a (first or second) conjugate-binding protein (see below). The (first or second) conjugate-binding protein is fixed within the strip (detection band), preferably along a band (detection band) positioned substantially perpendicular to the direction of sample flow along the strip. Thus, when the sample flow reaches the detection band along with the (1- or 2-)AAD-protein antibody conjugate recruited in the sample, it comes into contact with (or at least some of the AAD-protein antibody conjugate binds to) the first conjugate-binding protein contained in the detection band. This indicates the presence of the (1- or 2-)AAD-protein in the sample by a visible color change. This detection band can be composed of polymers or paper, such as sintered polymers, porous paper, microstructured polymers, or nitrocellulose.

[0052] In the context of this invention, the term “immobilized” is understood to mean, firstly, to be contained within the detection band and strip. Preferably, when the fluid comes into contact with the immobilized object, it means that the immobilized object is not mobilized, even if it continues to flow by capillary flow. Thus, when the immobilized object (e.g., an immobilized (first or second) conjugate-binding protein) reaches (and comes into contact with) the “immobilized” (first or second) conjugate-binding protein in the detection band (together with the mobilized (1- or 2-) AAD-protein antibody conjugate in the sample), it means that it is not moved by the fluid sample.

[0053] In the context of this invention, the terms “contact” or “contacts” mean that one entity comes into contact with another entity. It also includes “binding.” Therefore, “contact” also includes the case where a “conjugate-binding protein” binds to an (mobilized) “AAD-protein antibody conjugate” (e.g., “mobilized 1-AAD-protein antibody conjugate”).

[0054] In the context of this invention, the term “conjugate binding site” is understood to include two different embodiments: a) a “conjugate-binding protein” (see below), or b) a structure in which the surface structure (site) of a biomarker 1-AAD-protein, 2-AAD-protein, or X-AAD-protein is presented in a liquid, such as a sample. In this case, the conjugate binding site is immobilized within a strip and is usually immobilized on a detection band (usually the first detection band), but may also be immobilized on another (second) detection band. That is, in a sample containing a recruited 1-AAD-protein antibody conjugate (recruited 2-AAD-protein antibody conjugate), if the “protein antibody” has not yet bound to the biomarker / AAD-protein, the recruited 1-AAD-protein antibody conjugate (recruited 2-AAD-protein antibody conjugate) may bind to the conjugate-binding site.

[0055] In the context of the present invention, the term “conjugate-binding protein” is understood as a protein that binds to a conjugate containing an antibody. Preferably, the conjugate-binding protein binds to the “protein antibody” (above). The “conjugate-binding protein” binds to the “protein antibody” (above) either a) directly to the “protein antibody” (above) which is part of the “conjugate” (above), or b) indirectly to the “protein antibody” (above) by binding to a biomarker as an antigen (1-AAD-protein, 2-AAD-protein, or X-AAD-protein) that binds via the “protein antibody” of the “conjugate” (above). However, most importantly, the “conjugate-binding protein” binds only to the protein antibody of the conjugate, directly or indirectly, if the protein antibody of the conjugate is (preferably already) bound to the biomarker / AAD-protein. This is true in the preferred embodiment of the present invention in which the LFA is performed as a sandwich assay. It should be understood that the term “contact” also includes the words “binding” or “binding” in the context of “conjugate-binding protein”. The "conjugate-binding protein" is immobilized on a detection band (usually the first detection band) within the strip, but may also be immobilized on another (second) detection band. In a preferred embodiment, the "conjugate-binding protein" directly binds to the biomarker as an antigen that is also bound to the protein antibody of the conjugate, and the conjugate is recruited from the conjugate pad by a liquid from the sample to which the protein antibody of the conjugate is bound to the biomarker. In a very preferred embodiment, the "conjugate-binding protein" directly binds to HMGI-C that is also bound to the protein antibody of the conjugate, and the conjugate is recruited from the conjugate pad by a liquid from the sample to which the protein antibody of the conjugate is bound to HMGI-C. Alternatively, the "conjugate-binding protein" is an FC-binding protein, preferably a protein G, a protein A, or a protein A / G fusion protein. If the "conjugate-binding protein" binds to the same biomarker as the "protein antibody" (in the "conjugate"), the "conjugate-binding protein" can bind to the same epitope on the same biomarker as the "protein antibody" (in the "conjugate"), or the "conjugate-binding protein" can bind to a different epitope on the same biomarker as the "protein antibody" (in the "conjugate").

[0056] In the context of this invention, the term “further conjugate-binding protein” is understood to mean a protein that binds to a recruited MI-protein antibody sample conjugate, which includes an antibody against the MI-protein (see below). The further conjugate-binding protein is distinct from the first and second immobilized conjugate-binding proteins. The “further conjugate-binding protein” binds to the “MI-protein antibody” (described below) either a) directly to the “MI-protein antibody” (described below), which is part of the conjugate, or b) indirectly to the “MI-protein antibody” (described below) by binding to the MI-protein, 2-AAD-protein, or X-AAD-protein that binds via the “MI-protein antibody” being conjugated. The “further conjugate-binding protein” binds to the conjugated MI-protein antibody, directly or indirectly, only if the conjugated MI-protein antibody is bound to the MI-protein.

[0057] In the context of the present invention, the term “presence or absence of color” is understood to mean either a) a “visible color change” of the detection band, or b) no color change of the detection band, when the recruited (1- or 2-)AAD-protein antibody conjugate in the sample comes into contact with the (1st or 2nd) conjugate-binding protein in the (1st or 2nd) detection band.

[0058] In the context of the present invention, “visible color change” means a color change in the detection band that is (usually) detectable with the naked eye when the recruited (1- or 2-)AAD-protein antibody conjugate in the sample comes into contact with the (1st or 2nd) conjugate-binding protein in the (1st or 2nd) detection band. For example, if the “conjugate” contains gold particles, this would be a color change from white to red, or if the “conjugate” contains blue latex particles, this would be a color change from white to blue.

[0059] In the context of the present invention, the term "DISAMIN" is an acronym encompassing D-dimer, smooth muscle actin, myosin isozyme CK-MB, and nucleic acid components. Therefore, the AAD biomarker group represented by "DISAMIN" includes D-dimer, smooth muscle actin, myosin isozyme CK-MB, and nucleic acid components, and in particular includes "D-dimer," "smooth muscle actin," "myosin heavy chain," and "isozyme CK-MB."

[0060] In the context of this invention, the term "D-dimer" is understood as a specific degradation product of cross-linked fibrin, typically produced by plasmin hydrolysis. D-dimer measurements in the heart are used to detect pulmonary embolism and deep vein thrombosis.

[0061] In the context of this invention, the term "smooth muscle actin" is understood as actin protein, which is a spherical, multifunctional protein that forms microfilaments and is involved in the contractile organs of smooth muscle.

[0062] In the context of this invention, the term "smooth muscle myosin heavy chain" is understood to mean a heavy chain of myosin derived from smooth muscle cells.

[0063] In the context of this invention, the term "cardiac myosin light chain" is understood to mean a light chain of myosin derived from cardiomyocytes.

[0064] In the context of the present invention, the term "isozyme CK-MB" means that the creatine kinase myocardial band is a conjugate of two variants of the creatine phosphate kinase enzyme (isozyme CKM and CKB). More precisely, in the present invention, the term "isozyme CK-MB" means a creatine kinase myocardial band that is a conjugate of two variants of the creatine phosphate kinase enzyme (isozyme CKM and CKB).

[0065] In the context of the present invention, the term "creatinine phosphokinase (CK) or its MB isozyme" (also known as CPK) is synonymous with creatine kinase (CK), and MB means "myocardial band." More precisely, in the present invention, the term "creatinine phosphokinase (CK) or its MB isozyme" (also known as CPK) is synonymous with creatine kinase (CK), and MB means "myocardial band." In the context of this invention, the term “troponin T (TnT)” is understood to be a part of troponin (or the troponin complex). Troponin T is essential for the contraction of skeletal and cardiac muscle, but not for the contraction of smooth muscle. More precisely, in this invention, the term “troponin T (TnT)” is understood to be a part of troponin (or the troponin complex). It is essential for the contraction of skeletal and cardiac muscle, but not for the contraction of smooth muscle. In the context of this invention, the term "HMGI-C" (also known as HMGA2) is understood as a protein belonging to the non-histone chromosome high mobility group (HMG) protein family. HMG proteins function as builders and are essential components of enhanceosomes. They may function as transcription regulators, and their expression in adult tissues is generally associated with the formation of both malignant and benign tumors. More precisely, in the context of this invention, the term "HMGI-C" (also known as HMGA2) is understood as a protein belonging to the non-histone chromosome high mobility group (HMG) protein family. HMG proteins function as builders and are essential components of enhanceosomes. They may function as transcription regulators, and their expression in adult tissues is generally associated with the formation of both malignant and benign tumors.

[0066] In the context of this invention, the term "aggrecan" is understood as a proteoglycan capable of forming large aggregates in cartilage tissue. Aggrecan belongs to the chondroitin sulfate proteoglycan family and is an essential component of the extracellular matrix of cartilage tissue.

[0067] In the context of this invention, the term "soluble aortic elastin" refers to soluble elastin present in the aorta. Elastin is an important component of the extracellular matrix. It possesses high elasticity, is present in connective tissue, and allows many tissues in the body to return to their original shape after stretching or contracting. Elastin is used in areas where mechanical energy needs to be stored.

[0068] In the context of this invention, the term "elastin degradation product" is understood to mean the degradation product of elastin.

[0069] In the context of this invention, the term "human cardiac fatty acid-binding protein" (hFABP) is also known as mammary gland-derived growth inhibitor and is a small cytoplasmic protein released from cardiomyocytes after an ischemic attack. hFABP is involved in active fatty acid metabolism, transporting fatty acids from the cell membrane to mitochondria and promoting oxidation reactions.

[0070] In the context of this invention, the term "acute myocardial infarction" refers to a medical complication in which blood flow to the coronary arteries of the heart is reduced or stopped, causing damage to the heart muscle. It is also commonly known as a heart attack.

[0071] In the context of this invention, the term "MI-protein" refers to a protein that serves as an indicator of acute myocardial infarction. Examples include creatinine phosphokinase (CK) or its MB isozyme, cardiac myosin light chain, troponin T (TnT), and human cardiac fatty acid-binding protein (MI-protein).

[0072] In the context of this invention, the term “MI detection band” is understood as a region on a strip through which a fluid sample or analyte and a recruited MI-protein antibody (sample) conjugate are transported by capillary action. The MI detection band is positioned substantially perpendicular to the direction of sample flow along the strip and is typically spaced apart from the (first or second) detection band. The MI detection band contains further conjugate-binding proteins (see below). The further conjugate-binding proteins are fixed within the strip (MI detection band), preferably along a band (MI detection band) positioned substantially perpendicular to the direction of sample flow along the strip. Thus, when the sample flow reaches the MI detection band along with the recruited MI-protein antibodies bound in the sample, it comes into contact with the further binding proteins contained in the MI detection band (or at least some of the MI-protein antibody conjugates bind to it). This indicates the presence of MI-protein in the sample by a visible color change. This detection band can be composed of polymers or paper, such as sintered polymers, porous paper, microstructured polymers, or nitrocellulose.

[0073] In the context of this invention, the term "wicking pad" is understood, for example, within a strip, as an area on (or part of) the strip to which a liquid sample or analyte is transported by capillary action. This is located at the opposite end of the strip (membrane) from the sample pad. It holds the liquid (sample or analyte) and reagents that have passed through the membrane (strip) by capillary action and functions more or less as a waste container. This wicking pad may be made of polymers or paper such as sintered polymers, porous paper, microstructured polymers, or nitrocellulose.

[0074] In the context of this invention, the terms "protein G, protein A, or protein A / G fusion protein" are understood as a group of proteins that bind to (particularly human) IgG antibodies, polyclonal antibodies, or monoclonal antibodies, as well as IgA, IgE, IgM, and IgD. Protein A / G is a fusion protein that combines the IgG-binding domains of both protein A and protein G.

[0075] Lateral flow devices and kits Referring to the drawings, Figure 1 shows an example of a lateral flow device (1) according to the present invention. The device (1) comprises a sample pad (2), a conjugate pad (3) containing a mobile conjugate containing an antibody protein conjugated to a detection agent and binding to the AAD-protein, a membrane (4), and an optional wicking pad (7) for receiving and holding fluid that has passed from the sample pad (2) through the conjugate pad (3) and membrane (4) by capillary flow. A detection band (5) containing an immobilized binding protein is shown. Band (6) is an optional positive control band. The device (1) may further include a backing (8).

[0076] In one embodiment, the present invention provides a lateral flow device for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian body fluid sample. The device comprises the following components: i) A strip formed with a material that allows capillary flow along a portion of the strip; ii) A sample pad positioned near one end of the strip for receiving a bodily fluid sample; iii) A first conjugate pad positioned within the strip, wherein during operation, the sample flows through the strip by capillary action from the sample pad to the conjugate pad, mobilizing the conjugate contained in the conjugate pad, the conjugate containing a protein antibody conjugated to the detection agent and bound to the 1-AAD-protein, iv) A first detection band comprising a first conjugate-binding protein fixed within the strip along a band positioned substantially perpendicular to the direction of sample flow within the strip, wherein when a recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the first conjugate-binding protein in the first detection band, the presence of the 1-AAD-protein in the sample is indicated by a visible color change, Here, the 1-AAD-protein is selected from a group of AAD biomarkers including D-dimers, smooth muscle actin myosin isozymes, CK-MB, and nucleic acid components (DISAMIN), particularly HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin.

[0077] In a preferred embodiment A of the lateral flow device according to the present invention, the 1-AAD-protein is aggrecan.

[0078] In another alternative preferred embodiment A of the lateral flow device according to the present invention, the 1-AAD-protein is HMGI-C.

[0079] In these preferred embodiments A, the lateral flow device according to the present invention binds the protein antibody to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1.

[0080] In a preferred embodiment B of the lateral flow device according to the present invention, the 1-AAD-protein is HMGI-C, and the lateral flow device further comprises a second protein antibody that binds to a second protein (2-AAD-protein) that may be released during an event causing acute aortic dissection, preferably the protein antibody that binds to the 1-AAD-protein binds to an antigen of the amino acid sequence or fragment thereof of SEQ ID NO: 1. Preferably, the 2-AAD-protein is selected from a group of AAD biomarkers including D-dimers, smooth muscle actin myosin isozyme CK-MB, and nucleic acid components (DISAMIN), and is particularly selected from aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, or D-dimer. Even more preferably, the 2-AAD-protein is a D-dimer.

[0081] Most preferably, in this embodiment B, the 1-AAD-protein is HMGI-C and the 2-AAD-protein is a D-dimer.

[0082] This is a highly preferred embodiment of the lateral flow device of the present invention, comprising at least a first antibody against HMGI-C (1-AAD-protein) and a second antibody that binds to the D-dimer (X-AAD-protein). This combination enables very good differentiation between AAD and MI, and the combination of HMGI-C and D-dimer significantly improves the detection accuracy of AAD.

[0083] In Example B of the preceding paragraph, it is preferable that in the lateral flow device according to the present invention, the second protein antibody against the 2-AAD protein is bound to a second detection agent different from the one bound to the antibody against the 1-AAD protein, and the conjugate of the second protein antibody against the 2-AAD protein is located on either the first binding pad or a further binding pad. The second protein antibody against the 2-AAD-protein is bound to the same detection agent as the antibody against the 1-AAD-protein, and the conjugate of the second protein antibody against the 2-AAD-protein is positioned on either the first binding pad or a further binding pad, preferably such that the first conjugate-binding protein in the first detection band comes into contact with the first conjugate-binding protein in the first detection band, indicating the presence of the 2-AAD-protein in the sample by a visible color change.

[0084] In these embodiments B of the preceding paragraph, it is preferable that the lateral flow device according to the present invention comprises a second detection band containing a second conjugate-binding protein distinct from the first, immobilized within the strip along a band located away from the first detection band and substantially perpendicular to the direction of sample flow along the strip, and when a recruited 2-AAD-protein antibody conjugate in the sample comes into contact with the second conjugate-binding protein in the second detection band, the presence of the 2-AAD-protein in the sample is indicated by a visible color change.

[0085] In a preferred embodiment C of the lateral flow device according to the present invention, the lateral flow device further comprises one or more further protein antibodies that bind to one or more further proteins potentially released during the event leading to acute aortic dissection (X-AAD-protein), preferably the 1-AAD-protein and X-AAD protein are selected from a group of AAD biomarkers including the D-dimer smooth muscle actin myosin isozyme CK-MB and nucleic acid components (DISAMIN), particularly aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, or D-dimer.

[0086] In a preferred embodiment D of the lateral flow device according to the present invention, the 1-AAD-protein is HMGI-C, and the lateral flow device further comprises one or more further protein antibodies that bind to one or more further proteins (X-AAD-proteins) potentially released during an event causing acute aortic dissection, preferably the protein antibodies are bound to an antigen or fragment thereof of the amino acid sequence of SEQ ID NO: 1, and preferably the X-AAD-protein is selected from a group of AAD biomarkers including D-dimers, smooth muscle actin, myosin isozyme CK-MB, and nucleic acid components (DISAMIN), particularly aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, or D-dimer.

[0087] In preferred embodiments C or D of the lateral flow device according to the present invention, the one or more additional protein antibodies against the X-AAD- protein are bound to the same detection agent as those bound to the antibody against the 1-AAD- protein, the conjugates of the one or more additional protein antibodies against the X-AAD- protein are located on the first binding pad, and the first conjugate-binding protein of the first detection band indicates the presence of one or more X-AAD- proteins in the sample by a visible color change when one or more further recruited X-AAD- protein antibody conjugates in the sample come into contact with the first conjugate-binding protein of the first detection band.

[0088] A general principle of the lateral flow device of this invention is the need to identify proteins that show different elevated blood (including serum / plasma) concentrations in patients with aortic dissection and patients with myocardial infarction. Notably, myocardial infarction (MI) and acute myocardial infarction (AAD) often present with the same symptoms, but to date, the gold standard for MI diagnosis is based on "STE" in 12-lead electrocardiogram diagnosis.

[0089] In another (and therefore preferred) embodiment of the lateral flow device according to the present invention, the lateral flow device further comprises at least one MI-protein antibody that binds to an indicator protein of acute myocardial infarction (MI-protein), and another MI-detection band containing a further conjugate-binding protein different from first and second immobilized conjugate-binding proteins immobilized in the strip, the band being positioned substantially perpendicular to the direction of sample flow along the strip, so that when at least one recruited MI-protein antibody sample conjugate in the sample comes into contact with the further conjugate-binding protein in the MI-detection band, the presence of the MI-protein in the sample is indicated by a visible color change, preferably the MI-protein is selected from the group consisting of creatinine phosphokinase (CK) or its MB isozyme, cardiac myosin light chain, troponin T (TnT) (or troponin I (TnI)), and human cardiac fatty acid-binding proteins.

[0090] In this embodiment, the MI protein (to which the MI protein antibody binds) is very preferably troponin T (TnT) or troponin I (ThI), or troponin T (TnT). Since MI and AAD often present with the same symptoms, this allows for good differentiation between AAD and MI.

[0091] Furthermore, in this embodiment, the MI-protein (to which the MI-protein antibody binds) is preferably troponin T (TnT) or troponin I (ThI), or troponin T (TnT), and the other MI-protein is preferably creatinine phosphokinase (CK), particularly its MB isozyme (CK-MB). This combination enables very good identification of MI, and the combination of troponin and CK-MB improves the accuracy of detecting myocardial infarction (e.g., over 91%). Also, since MI and AAD often present with the same symptoms, good differentiation between AAD and MI is possible.

[0092] In this embodiment, most preferably, the lateral flow device according to this embodiment is defined by the following: - The lateral flow device further includes an MI-protein antibody that binds to an MI-protein that is either troponin T-like protein (TnT) or troponin I (ThI), or troponin T (TnT); - 1-AAD-protein is HMGI-C; and, - The lateral flow device further includes another protein antibody that binds to another protein (X-AAD-protein) that is potentially released during events causing acute aortic dissection, where X-AAD-protein is a D-dimer.

[0093] Therefore, the lateral flow device consists of a primary antibody against HMGI-C (1-AAD-protein), a secondary antibody that binds to troponin (MI-protein), and a tertiary antibody that binds to D-dimer (X-AAD-protein). This combination allows for very good differentiation between AAD and MI, and the combination of HMGI-C and D-dimer significantly improves the detection accuracy of AAD.

[0094] Furthermore, most preferably in this embodiment, the lateral flow device according to this embodiment is defined by the following: - The lateral flow device further contains two MI-protein antibodies, one antibody which binds to the MI-protein troponin T (TnT) or troponin I (ThI) (especially troponin T (TnT)), and the other antibody which binds to the MI-protein creatinine phosphokinase (CK), especially its MB isozyme (CK-MB); - The I-AAD protein is HMGI-C; and - The lateral flow device further includes another protein antibody that binds to another protein (X-AAD-protein) that may be released during the event leading to acute aortic dissection, where X-AAD-protein is a D-dimer.

[0095] Therefore, the lateral flow device of the present invention includes a first antibody against HMGI-C (I-AAD-protein), a second antibody that binds to troponin (MI-protein), a third antibody that binds to D-dimer (X-AAD-protein), and a fourth antibody that binds to CK, particularly its MB isozyme (CK-MB). This combination enables very good differentiation between AAD and MI, the combination of HMGI-C and D-dimer significantly improves the detection accuracy of AAD, and the combination of troponin and CK-MB improves the detection accuracy of myocardial infarction (e.g., over 91%).

[0096] In another preferred embodiment of the lateral flow device according to the present invention, acute aortic dissection (AAD) is an aortic dissection including acute type A aortic dissection, and / or an acute aortic syndrome including one or more of the following aortic dissections / acute aortic syndromes: - Perforated aortic ulcer and intramural hematoma - Thoracic aortic aneurysm - Chronic dissociation - Indications for treatment with branched and windowed stent grafts - Various treatment options for aortic arch dissection with Comerel's diverticulum - Secondary interventions after endovascular repair of aortic dissection - Pseudoaneurysm complicated with internal carotid artery dissection - Established indications for invasive treatment of thoracic aortic aneurysms - Aortic arch dissection: Controversy surrounding classification - Treatment algorithm for patients with (sub)acute type B aortic dissection - Thoracic descending aortic dissection - Differences in aortic disease in bicuspid valve patients, regardless of the presence or absence of aortic coarctation. - Angina pectoris, chest pain - Dyspnea accompanied by hypertensive crisis - Sudden coma, or, - Shock of unknown cause.

[0097] Another preferred embodiment of the lateral flow device according to the present invention further comprises a wicking pad for receiving and holding the sample after it has passed through the detection band.

[0098] In another preferred embodiment of the lateral flow device according to the present invention, the conjugate-binding protein is an FC-binding protein, preferably protein G, protein A, or protein A / G fusion protein.

[0099] In another preferred embodiment of the lateral flow device according to the present invention, the conjugate-binding protein binds to the same biomarker as the protein antibody against the AAD-protein ("conjugate"). The "conjugate-binding protein" may bind to the same epitope on the same biomarker as the "protein antibody" ("conjugate"), or it may bind to a different epitope on the same biomarker as the "protein antibody" ("conjugate").

[0100] In another preferred embodiment of the lateral flow device according to the present invention, the conjugate-binding protein binds to the same epitope on the same biomarker as the "protein antibody" (in "conjugate").

[0101] In another preferred embodiment of the lateral flow device according to the present invention, the “conjugate-binding protein” can bind to an epitope on the same biomarker that is different from the epitope on the same biomarker to which the “protein antibody” (in the “conjugate”) binds.

[0102] In another preferred embodiment of the lateral flow device according to the present invention, the antibody-bound detection agent is selected from the group comprising metal nanoparticles or nanoshells, non-metallic nanoparticles or nanoshells, enzymes, and fluorescent molecules.

[0103] In another preferred embodiment of the lateral flow device according to the present invention, the antibody-bound detection agent comprises metallic gold nanoparticles or nanoshells.

[0104] In another preferred embodiment of the lateral flow device according to the present invention, the sample pad comprises a filter membrane for removing one or more components from the sample.

[0105] In another preferred embodiment of the lateral flow device according to the present invention, one or more components removed from the sample by the filter membrane pad are cells, cellular material, fats, or particulate matter.

[0106] Another preferred embodiment of the lateral flow device according to the present invention further comprises a control line positioned substantially perpendicular to the direction of sample flow along the strip. Preferably, the conjugate pad further comprises immunoglobulin conjugated to a detection agent, and during operation, the sample flows from the sample pad to the conjugate pad, mobilizing the immunoglobulin conjugate so that the immunoglobulin conjugate passes through the detection band without reacting and crosses the control line. Preferably, the control line is coated with an antibody to which the mobilized immunoglobulin conjugate can bind as it crosses the control line, and optionally, the binding at the control line is indicated by a visible color change.

[0107] In another preferred embodiment of the lateral flow device according to the present invention as described in the previous paragraph, the immunoglobulin in the conjugate is derived from an animal species other than the mammalian species from which the body fluid sample originates, and / or the detection agent conjugated to the immunoglobulin is selected from the group including metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules, preferably the detection agent conjugated to the immunoglobulin includes metallic gold nanoparticles or nanoshells.

[0108] In another preferred embodiment of the lateral flow device according to the present invention, the strip is formed from nitrocellulose.

[0109] In another preferred embodiment of the lateral flow device according to the present invention, the bodily fluid sample is from a human, particularly a patient.

[0110] In another preferred embodiment of the lateral flow device according to the present invention, the body fluid is selected from the group including whole blood, serum, and plasma.

[0111] A further aspect of the present invention also includes a kit for detecting a protein (AAD-protein) released during an event leading to acute aortic dissection in a mammalian body fluid sample, the kit comprising: c) The above-mentioned lateral flow assay device; and, d) Instructions on how to use the lateral flow assay device described above.

[0112] method A further aspect of the present invention includes a method for detecting the AAD-protein in mammalian body fluids, which involves using the lateral flow assay device described above.

[0113] The present invention also includes a method for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian body fluid sample, the method comprising the following steps: c) A step of placing a sample on a sample pad of a lateral flow assay device, comprising the following steps: (1) A strip formed of a material that allows capillary flow along a portion of the strip; (2) A sample pad positioned near one end of the strip to receive a bodily fluid sample; (3) A first conjugate pad positioned within the strip, wherein during operation, the sample flows through the strip by capillary action from the sample pad to the conjugate pad, mobilizing the conjugate contained in the conjugate pad, the conjugate containing a protein antibody conjugated to the detection agent and bound to the 1-AAD-protein, (4) A first detection band comprising a first conjugate-binding protein fixed along a band positioned substantially perpendicular to the direction of sample flow along the strip, wherein when a recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the first conjugate-binding protein in the first detection band, the presence of the 1-AAD-protein in the sample is indicated by a visible color change. Here, the 1-AAD-protein is selected from a group of AAD biomarkers including D-dimer, smooth muscle actin myosin isozyme, CK-MB, and nucleic acid component (DISAMIN), and is particularly selected from HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, preferably HMGI-C, and the antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1. d) The presence of labeled particles in the detection region is examined to detect whether the AAD-protein is present in the sample.

[0114] The present invention also includes a method for determining acute aortic dissection (AAD) in a patient by immunological measurement, the method comprising: d) Take a sample of the patient's bodily fluids. a. At least one antibody against the first protein (1-AAD-protein) released during the event leading to acute aortic dissection, and b. Incubate with binding partner B for either the 1-AAD- protein or the antibody, Here, either the antibody or the binding partner B is bound to the detection agent. It forms an immunological complex containing the detection agent; e) Isolate the immunological complex from the remaining sample; and, f) Determine the presence of the detection agent in the isolated complex or in the remaining sample as an indicator of the 1-AAD- protein in the sample; This determines the occurrence of AAD in the patient. Here, the 1-AAD-protein is selected from a group of AAD biomarkers including the D-dimer smooth muscle actin myosin isozyme CK-MB and nucleic acid components (DISAMIN), particularly HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, preferably HMGI-C, and the antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1.

[0115] The present invention will be described in more detail based on embodiments. It will be understood that the present invention as described in the claims is not limited in any way by these embodiments.

[0116] Embodiments: 1. A lateral flow device for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian body fluid sample, comprising the following components: a) A strip formed of a material that allows fluid flow by capillary action along a portion of the strip; ii) A sample pad positioned near one end of the strip to receive a bodily fluid sample; iii) A first conjugate pad positioned within the strip, wherein during operation, the sample flows through the strip by capillary action from the sample pad to the conjugate pad, mobilizing the conjugate contained in the conjugate pad, the conjugate containing a protein antibody conjugated to the detection agent and bound to the 1-AAD-protein, iv) A first detection band comprising a conjugate binding site fixed within the strip along a band positioned substantially perpendicular to the direction of sample flow along the strip, wherein when a recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the conjugate binding site of the first detection band, the presence or absence of the 1-AAD-protein in the sample is indicated by the presence or absence of color, Here, the 1-AAD- protein was selected from a group of AAD biomarkers including D-dimer, smooth muscle actin, myosin, isozyme CK-MB, and nucleic acid component (DISAMIN), in particular HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin. Preferably, the presence or absence of color is a visible color change, and the conjugate binding site is the first conjugate-binding protein. Alternatively, the conjugate binding site is a surface structure (site) of the biomarker 1-AAD- protein, which is presented on the recruited 1-AAD- protein antibody conjugate and can bind even if the protein antibody has not yet bound.

[0117] 1a. A lateral flow device for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian body fluid sample, comprising the following components: a) A strip formed of a material that allows capillary flow along a portion of the strip; ii) A sample pad positioned near one end of the strip to receive a bodily fluid sample; iii) A first conjugate pad positioned within the strip, wherein during operation, the sample flows through the strip by capillary action from the sample pad to the conjugate pad, mobilizing the conjugate contained in the conjugate pad, the conjugate containing a protein antibody conjugated to the detection agent and bound to the 1-AAD-protein, iv) A first detection band comprising a conjugate binding site fixed within the strip along a band positioned substantially perpendicular to the direction of sample flow along the strip, wherein when a recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the conjugate binding site of the first detection band, the presence or absence of the 1-AAD-protein in the sample is indicated by the presence or absence of color, Here, the 1-AAD-protein is selected from a group of AAD biomarkers, particularly HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, and D-dimer, and comprises smooth muscle actin, myosin, isozyme CK-MB, and nucleic acid component (DISAMIN). Preferably, the presence or absence of color is a visible color change, and the conjugate binding site is the first conjugate-binding protein. Alternatively, The conjugate binding site is a surface structure (site) of the biomarker 1-AAD- protein, which is presented on the recruited 1-AAD-protein antibody conjugate and can bind even if the protein antibody has not yet bound.

[0118] 2. A lateral flow device according to Embodiment 1 or 1a, wherein the 1-AAD-protein is aggrecan.

[0119] 3. A lateral flow device according to Embodiment 1 or 1a, wherein the 1-AAD-protein is HMGI-C.

[0120] 3a. A lateral flow device according to Embodiment 1 or 1a, wherein the 1-AAD-protein is a D-dimer.

[0121] 4. A lateral flow device according to Embodiment 1 or 1a, wherein the protein antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1.

[0122] 5. A lateral flow device according to Embodiment 1 or 1a, wherein the 1-AAD-protein is HMGI-C, and the lateral flow device further comprises a second protein antibody that binds to a second protein (2-AAD-protein) potentially released during the event leading to acute aortic dissection, preferably the protein antibody that binds to the 1-AAD-protein binds to an antigen or fragment thereof of the amino acid sequence of SEQ ID NO: 1.

[0123] 6. A lateral flow device according to Embodiment 5, wherein the 2-AAD-protein is selected from the group of AAD biomarkers including D-dimer, smooth muscle actin myosin isozyme CK-MB, and nucleic acid component (DISAMIN), particularly from aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, or D-dimer, preferably D-dimer, most preferably the 1-AAD-protein is HMGI-C and the 2-AAD-protein is D-dimer.

[0124] 7. A lateral flow device according to any one of Embodiments 5 or 6, wherein the second protein antibody against the 2-AAD protein is bound to a second detection agent different from the one bound to the antibody against the 1-AAD protein, and the conjugate of the second protein antibody against the 2-AAD protein is located on either the first binding pad or a further binding pad.

[0125] 8. A lateral flow device according to any one of Embodiments 5 or 6, wherein the second protein antibody against the 2-AAD-protein is bound to the same detection agent as the antibody against the 1-AAD-protein, and the conjugate of the second protein antibody against the 2-AAD-protein is located on either the first binding pad or a further binding pad.

[0126] 9. A lateral flow device according to Embodiment 8, wherein the first conjugate-binding protein of the first detection band is such that when a second mobile 2-AAD-protein antibody conjugate in the sample comes into contact with the first conjugate-binding protein of the first detection band, the presence of 2-AAD-protein in the sample is indicated by a visible color change.

[0127] 10. Lateral flow device according to any one of Embodiments 7 or 8. A lateral flow device comprising a second detection band containing a second conjugate-binding protein distinct from a first conjugate-binding protein immobilized in a strip along a band located away from a first detection band, wherein the band is substantially perpendicular to the direction of sample flow along the strip, and the presence of the 2-AAD-protein in the sample is indicated by a visible color change when a recruited 2-AAD-protein antibody conjugate in the sample comes into contact with the second conjugate-binding protein in the second detection band.

[0128] 11. A lateral flow device according to Embodiment 1 or 1a, wherein the lateral flow device further comprises one or more additional protein antibodies that bind to one or more additional proteins (X-AAD-proteins) potentially released during an event causing acute aortic dissection.

[0129] 12. A lateral flow device according to Embodiment 11, wherein the 1-AAD-protein and X-AAD protein are selected from a group of AAD biomarkers including D-dimer, smooth muscle actin myosin isozyme CK-MB, and nucleic acid components (DISAMIN), and are particularly selected from aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, or D-dimer.

[0130] 13. A lateral flow device according to Embodiment 1 or 1a, wherein the 1-AAD-protein is HMGI-C, and the lateral flow device further comprises one or more further protein antibodies that bind to one or more further proteins (X-AAD-proteins) potentially released during an event causing acute aortic dissection, preferably the protein antibodies bind to an antigen or fragment thereof of the amino acid sequence of SEQ ID NO: 1.

[0131] 14. A lateral flow device according to Embodiment 13, wherein the X-AAD-protein is selected from a group of AAD biomarkers including D-dimer, smooth muscle actin, myosin isozyme CK-MB, and nucleic acid component (DISAMIN), particularly from aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, or D-dimer, Here, the X-AAD protein is the D-dimer and isozyme CK-MB, or Here, the X-AAD protein is a D-dimer in a lateral flow device.

[0132] 14a. A lateral flow device according to Embodiment 14, wherein the 1-AAD-protein is HMGI-C and the X-AAD-protein is a D-dimer and isozyme CK-MB.

[0133] 14b. A lateral flow device according to Embodiment 14, wherein the 1-AAD protein is HMGI-C and the X-AAD protein is a D-dimer.

[0134] 15. A lateral flow device according to any one of embodiments 11 to 14b, wherein the one or more additional protein antibodies against the X-AAD-protein are bound to the same detection agent as those bound to the antibody against the 1-AAD-protein, the conjugate of the one or more additional protein antibodies against the X-AAD-protein is located on the first conjugate pad, and the presence of the one or more X-AAD-protein in the sample is indicated by a visible color change when the first conjugate-binding protein in the first detection band comes into contact with the first conjugate-binding protein in the sample.

[0135] 16. A lateral flow device according to any one of the embodiments, further comprising at least one MI-protein antibody that binds to an indicator protein of acute myocardial infarction (MI-protein), and another MI detection band containing a further conjugate-binding protein different from first and second immobilized conjugate-binding proteins immobilized in a strip, wherein the band is positioned substantially perpendicular to the direction of sample flow along the strip, so that when at least one mobilized MI-protein antibody sample conjugate in the sample comes into contact with the further conjugate-binding protein in the MI-detection band, the presence of the MI-protein in the sample is indicated by a visible color change.

[0136] 17. A lateral flow device according to Embodiment 16, wherein the MI-protein is selected from the group consisting of creatinine phosphokinase (CK) or its MB isozyme, cardiac myosin light chain, troponin T (TnT), and human cardiac fatty acid-binding protein.

[0137] 17a. A lateral flow device according to Embodiment 16, wherein the MI-protein is selected from the group consisting of creatinine phosphokinase (CK) or its MB isozyme, cardiac myosin light chain, troponin (such as troponin T (TnT) or troponin I (TnI)), and human cardiac fatty acid-binding protein.

[0138] 17b. A lateral flow device according to Embodiment 16, further comprising an MI-protein antibody that binds to a troponin T-like protein (TnT) or troponin I (ThI), or an MI-protein that is troponin T (TnT). [This is a highly preferred embodiment of the lateral flow device of the present invention, comprising at least a first antibody against HMGI-C (1-AAD-protein) and a second antibody that binds to troponin (MI-protein). This combination allows for good differentiation between AAD and MI.]

[0139] 17c. A lateral flow device according to Embodiment 16, further comprising two MI-protein antibodies, one antibody bound to the MI-protein troponin T (TnT) or troponin I (ThI) (in particular troponin T (TnT)), and the other antibody bound to the MI-protein creatinine phosphokinase (CK), in particular its MB isozyme (CK-MB). [This is a highly preferred embodiment of the lateral flow device of the present invention, comprising at least a first antibody against HMGI-C (1-AAD protein), a second antibody bound to troponin (MI-protein), and a third antibody bound to CK, particularly its MB isozyme (CK-MB). This combination enables very good differentiation between AAD and MI, and the combination of troponin and CK-MB improves the accuracy of myocardial infarction detection (e.g., over 91%).]

[0140] 17d. A lateral flow device according to Embodiment 16, - The lateral flow device further comprises a troponin T-like protein (TnT) or troponin I (ThI), or an MI-protein antibody that binds to troponin T (TnT), - 1-AAD- protein is HMGI-C, - The lateral flow device further comprises another protein antibody that binds to another protein (X-AAD-protein) potentially released during events leading to acute aortic dissection, where X-AAD-protein is a D-dimer. Lateral flow device. [This is one of the most preferred embodiments of the lateral flow device of the present invention, comprising a first antibody against HMGI-C (1-AAD-protein), a second antibody bound to troponin (MI-protein), and a third antibody bound to D-dimer (X-AAD-protein). This combination enables very good differentiation between AAD and MI, and the combination of HMGI-C and D-dimer significantly improves the detection accuracy of AAD.]

[0141] 17e. A lateral flow device according to Embodiment 16, - The lateral flow device further contains two MI-protein antibodies, one antibody which binds to the MI-protein troponin T (TnT) or troponin I (ThI) (especially troponin T (TnT)), and the other antibody which binds to the MI-protein creatinine phosphokinase (CK), especially its MB isozyme (CK-MB); - The aforementioned 1-AAD-protein is HMGI-C; and, - The lateral flow device further includes another protein antibody that binds to another protein (X-AAD-protein) that may be released during an event leading to acute aortic dissection, and the X-AAD-protein is a D-dimer. Lateral flow device. [This is another most preferred embodiment of the lateral flow device of the present invention, comprising a first antibody against HMGI-C (1-AAD protein), a second antibody bound to troponin (MI-protein), a third antibody bound to D-dimer (X-AAD-protein), and a fourth antibody bound to CK, particularly its MB isozyme (CK-MB). This combination enables very good differentiation between AAD and MI, the combination of HMGI-C and D-dimer significantly improves the detection accuracy of AAD, and the combination of troponin and CK-MB improves the detection accuracy of myocardial infarction (e.g., over 91%).]

[0142] 18. A lateral flow device according to any of the above embodiments, wherein acute aortic dissection (AAD) is an aortic dissection including acute type A aortic dissection, and / or an acute aortic syndrome including one or more of the following aortic dissections / acute aortic syndromes: - Perforated aortic ulcer and intramural hematoma - Thoracic aortic aneurysm - Chronic dissociation - Indications for treatment with branched and windowed stent grafts - Various treatment options for aortic arch dissection with Comerel's diverticulum - Secondary interventions after endovascular repair of aortic dissection - Pseudoaneurysm complicated with internal carotid artery dissection - Established indications for invasive treatment of thoracic aortic aneurysms - Aortic arch dissection: Controversy surrounding classification - Treatment algorithm for patients with (sub)acute type B aortic dissection - Thoracic descending aortic dissection - Differences in aortic disease in bicuspid valve patients, regardless of the presence or absence of aortic coarctation. - Angina pectoris, chest pain - Dyspnea accompanied by hypertensive crisis - Sudden coma, or - Shock of unknown cause.

[0143] 19. The lateral flow device according to any one of the embodiments, further comprising a wicking pad for receiving and holding a sample after it has passed through the detection band.

[0144] 20. The lateral flow device according to any one of the embodiments, wherein the conjugate-binding protein is an FC-binding protein.

[0145] 21. A lateral flow device according to Embodiment 20, wherein the FC-binding protein is protein G, protein A, or protein A / G fusion protein.

[0146] 22. A lateral flow device according to any one of the embodiments, wherein the detection agent conjugated to the antibody is selected from the group comprising metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules.

[0147] 23. A lateral flow device according to any one of the embodiments, wherein the detection agent bound to the antibody comprises metallic gold nanoparticles or nanoshells.

[0148] 24. The lateral flow device according to any one of the embodiments, wherein the sample pad comprises a filter membrane for removing one or more components from the sample.

[0149] 25. The lateral flow device according to any one of the embodiments, wherein one or more components removed from the sample by the filter membrane pad are cells, cellular material, fat, or particulate matter.

[0150] 26. A lateral flow device according to any one of the embodiments described above, further comprising a control line positioned substantially perpendicular to the direction of sample flow along a strip.

[0151] 27. A lateral flow device according to Embodiment 26, wherein the conjugate pad further comprises immunoglobulin conjugated with a detection agent, and during operation, a sample flows from the sample pad to the conjugate pad, mobilizing an immunoglobulin conjugate that passes through the detection band without reaction and crosses the control line.

[0152] 28. A lateral flow device according to Embodiment 27, wherein an antibody capable of binding to an immunoglobulin conjugate mobilized as it crosses the control line is deposited on the control line, and optionally, the binding on the control line is indicated by a visible color change.

[0153] 29. A lateral flow device according to Embodiment 27 or 28, wherein the immunoglobulin in the conjugate is derived from an animal species other than the mammalian species from which the body fluid sample originates.

[0154] 30. A lateral flow device according to any one of embodiments 27 to 29, wherein the detection agent bound to immunoglobulin is selected from the group comprising metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules.

[0155] 31. A lateral flow device according to Embodiment 30, wherein the detection agent bound to immunoglobulin comprises metallic gold nanoparticles or nanoshells.

[0156] 32. A lateral flow device according to any of the above embodiments, wherein the strip is formed from nitrocellulose.

[0157] 33. A lateral flow device according to any one of the embodiments, wherein the bodily fluid sample is of human origin.

[0158] 34. A lateral flow device according to any one of the embodiments, wherein the body fluid is selected from the group comprising whole blood, serum, and plasma.

[0159] 35. A method for detecting the AAD protein in the body fluids of a mammal, comprising using the device described in any one of Embodiments 1 to 34.

[0160] 36. A method for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian fluid sample, comprising the following steps: e) The step of placing a sample on a sample pad of a lateral flow assay device; the lateral flow assay device includes: (1) A strip formed of a material that allows capillary flow along a portion of the strip; (2) A sample pad positioned near one end of the strip for receiving a bodily fluid sample; (3) A first conjugate pad positioned within the strip, wherein during operation, the sample flows through the strip by capillary action from the sample pad to the conjugate pad, mobilizing the conjugate contained in the conjugate pad, the conjugate containing a protein antibody conjugated to the detection agent and bound to the 1-AAD-protein, (4) A first detection band comprising a first conjugate-binding protein fixed along a band positioned substantially perpendicular to the direction of sample flow along the strip, wherein when a recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the first conjugate-binding protein in the first detection band, the presence of the 1-AAD protein in the sample is indicated by a visible color change. Here, the 1-AAD-protein is selected from a group of AAD biomarkers including D-dimers, smooth muscle actin myosin isozymes, CK-MB, and nucleic acid components (DISAMIN), and is particularly selected from HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, preferably HMGI-C, and the antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1; f) The presence of labeled particles in the detection region is examined to detect whether the AAD protein is present in the sample.

[0161] 37. A kit for detecting the protein (AAD-protein) released during events leading to acute aortic dissection in mammalian body fluid samples, comprising the following: e) A lateral flow assay device according to any one of Embodiments 1 to 34; and f) Instructions on how to use the device described in any one of Embodiments 1 to 34.

[0162] 38. A method for determining acute aortic dissection (AAD) in a patient by immunoassay, comprising the following steps: g) Take a sample of the patient's bodily fluids. a. At least one antibody against the first protein (1-AAD-protein) released during the event leading to acute aortic dissection, and b. Incubate with binding partner B for either the 1-AAD- protein or the antibody, Here, either the antibody or the binding partner B is bound to the detection agent. A step of forming an immunological complex containing a detection agent; h) The step of isolating the immunological complex from the remaining sample; and i) A step of measuring the detection agent in the isolated complex or the remaining sample as an indicator of the 1-AAD- protein in the sample; This determines the occurrence of AAD in the patient. Here, the 1-AAD-protein is selected from a group of AAD biomarkers including the D-dimer smooth muscle actin myosin isozyme CK-MB and nucleic acid components (DISAMIN), particularly HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, preferably HMGI-C, and the antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1.

[0163] The present invention will be described in more detail with reference to the following embodiments. It will be understood that the present invention as described in the claims is not limited in any way by these embodiments. [Examples]

[0164] Example 1 In Example 1, the antigen (biomarker) HMGI-C (high-mobility protein) was expressed, isolated, and purified. Recombinant protein antigen expression tests were then performed in E. coli and mammalian cells.

[0165] 1.a) Gene synthesis and subcloning into expression vectors HMGI cDNA tagged with various tags (His, His-SUMO, or MBP-His for bacterial expression; MBP-His for mammalian expression) was chemically synthesized with codon optimization to match the corresponding expression host, and then subcloned into expression vectors. The protein sequences are as follows, with a portion shown in Figure 5.

[0166] 1.b) Protein sequence Escherichia coli (EC) construct > HMGI-C-His-EC MSARGEGAGQPSTSAQGQPAAPAPQKRGRGPRKQQQEPTGEPSPKRPRGRPKGSKNKSPSKAAQKKAEATGEKRPRGPRKWAGVQWYNLGSLQPPPPRFKQFSCLRLLSSWDYRHPPPHPANFCIFSRDRVSPCWPPGWSRTPDLRGSHHHHHH Features: HMGI-C [1:147] His tag with linker [148:155]

[0167] >His-SUMO-HMGI-C-EC MGHHHHHHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGSSARGEGAGQPSTSAQGQPA APAPQKRGRGRPRKQQQEPTGEPSPKRPRGRPKGSKNKSPSKAAQKKAEATGEKRPRGRPRKWAGVQWYNLGSLQPPPPRFKQFSCLRLLSSWDYRHPPPHPANFCIFSRDRVSPCWPPGWSRTPDLR Features: His tag with linker [1:8] SUMO-tag:[9:106] Linker HMGI-C [108:253]

[0168] > MBP-HMGI-C-His-EC MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPAL DKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPN KELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQSGSSARGEGAGQPSTSAQGQ PAAPAPQKRGRGPRKQQQEPTGEPSPKRPRGRPKGSKNKSPSKAAQKKAEATGEKRPRGPRKWAGVQWYNLGSLQPPPPRFKQFSCLRLLSSWDYRHPPPHPANFCIFSRDRVSPCWPPGWSRTPDLRGSHHHHHH Features: MBP-tag:[1:383] TEV protease cleavage tag:[384:392] Linker HMGI-C [393:538] His tag with linker [540:547]

[0169] Mammalian cell (MC) construct > MBP-HMGI-C-His-MC MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPAL DKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPN KELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQSGSSARGEGAGQPSTSAQGQ PAAPAPQKRGRGPRKQQQEPTGEPSPKRPRGRPKGSKNKSPSKAAQKKAEATGEKRPRGPRKWAGVQWYNLGSLQPPPPRFKQFSCLRLLSSWDYRHPPPHPANFCIFSRDRVSPCWPPGWSRTPDLRGSHHHHHH Features: MBP-tag:[1:383] TEV protease cleavage tag:[384:392] Linker HMGI-C [393:538] His tag with linker [540:547]

[0170] 1.c) Expression and purification tests The protein was expressed in E. coli or HEK cells, and affinity purification for the 6His tag was performed using nickel resin. The results are as follows: -His tag and MBP tag constructs were successfully expressed in E. coli (Figure 6, Gel A and Gel B). - The SUMO-tagged construct was successfully expressed in E. coli, but its purity was lower than the other two constructs (Figure 6, Gel C). - Expression was not observed in mammalian cells (Figure 6, Gel D).

[0171] [Table 1]

[0172] Example 2 Antibodies that bind to the antigen expressed, isolated, and purified in Example 1 are generated.

Claims

1. A lateral flow device for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian fluid sample, the device comprising: i) A strip formed of a material that allows for capillary flow of fluid along a portion of the strip; ii) A sample pad positioned close to one end of the strip to receive a bodily fluid sample; iii) A first conjugate pad arranged on a strip, wherein during operation, the sample flows through the strip from the sample pad to the conjugate pad by capillary action, thereby mobilizing the conjugate contained in the conjugate pad, the conjugate comprising a protein antibody conjugated to the 1-AAD-protein, and the first conjugate pad iv) A first detection band consisting of the first conjugate-binding protein immobilized within the strip along a band substantially perpendicular to the flow direction of the sample along the strip, such that when the recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the first conjugate-binding protein in the first detection band, the presence of the 1-AAD-protein in the sample is indicated by a visible color change. Here, the 1-AAD-protein is selected from a group of biomarkers for acute aortic dissection (AAD), including HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, as well as D-dimer, smooth muscle actin myosin isozyme CK-MB, and nucleic acid component (DISAMIN).

2. The lateral flow device according to claim 1, wherein the 1-AAD-protein is aggrecan, or the 1-AAD-protein is HMGI-C, or the 1-AAD-protein is a D-dimer.

3. The lateral flow device according to claim 1 or 2, wherein the protein antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO:

1.

4. The lateral flow device according to claim 1, wherein the 1-AAD-protein is HMGI-C, and the lateral flow device further comprises one or more further protein antibodies that also bind to one or more further proteins (X-AAD-proteins) potentially released during the event leading to acute aortic dissection, preferably the protein antibody that binds to the 1-AAD-protein binds to an antigen or fragment thereof of the amino acid sequence of SEQ ID NO:

1.

5. A lateral flow device according to claim 4, wherein the X-AAD-protein is selected from a group of AAD biomarkers including D-dimer, smooth muscle actin, myosin, isoenzyme CK-MB, and nucleic acid component (DISAMIN), and is particularly selected from aggrecan, smooth muscle myosin heavy chain, soluble aortic elastin, or D-dimer, preferably D-dimer, or Here, the X-AAD-protein is a D-dimer and isozyme CK-MB, or Here, the X-AAD-protein is a D-dimer. Lateral flow device.

6. A lateral flow device according to claim 4 or 5, wherein the one or more further protein antibodies against the X-AAD-protein are bound to the same detection agent as those bound to the antibody against the 1-AAD-protein, the conjugate with the one or more further protein antibodies against the X-AAD-protein is located on the first binding pad, and the first conjugate-binding protein in the first detection band is indicated by a visible color change when the one or more further recruited X-AAD protein antibody conjugates in the sample come into contact with the first conjugate-binding protein in the first detection band.

7. A lateral flow device according to any one of claims 1 to 6, further comprising at least one MI-protein antibody that binds to an indicator protein of acute myocardial infarction (MI-protein), and a separate MI-detection band containing a further conjugate-binding protein different from first and second immobilized conjugate-binding proteins immobilized within a strip, wherein the band is positioned along a band substantially perpendicular to the direction of sample flow along the strip, and the presence of the MI-protein in the sample is indicated by a visible color change when at least one immobilized MI-protein antibody sample conjugate in the sample comes into contact with the further conjugate-binding protein in the MI-detection band, preferably the MI-protein is selected from the group consisting of creatinine phosphokinase (CK) or its MB isozyme, cardiac myosin light chain, troponin T (TnT) or troponin I (ThI), and human cardiac fatty acid-binding protein; Most preferably, the lateral flow device further includes an MI-protein antibody that binds to the MI-protein troponin (e.g., TnT) or troponin I (ThI); or Most preferably, the lateral flow device further includes two MI-protein antibodies that bind to the MI-protein troponin (e.g., TnT) or troponin I (ThI), and the other antibody binds to the MI-protein creatinine phosphokinase (CK), particularly its CK-MB isozyme.

8. Lateral flow device according to any one of the above claims, wherein acute aortic dissection (AAD) is an aortic dissection including acute type A aortic dissection, and / or an acute aortic syndrome including one or more of the following aortic dissection / acute aortic syndromes: - Perforated aortic ulcer and intramural hematoma - Thoracic aortic aneurysm - Chronic dissociation - Indications for treatment with branched and windowed stent grafts - Various treatment options for aortic arch dissection with Comerel's diverticulum - Secondary interventions after endovascular repair of aortic dissection - Pseudoaneurysm complicated with internal carotid artery dissection - Established indications for invasive treatment of thoracic aortic aneurysms - Aortic arch dissection: Controversy surrounding classification - Treatment algorithm for patients with (sub)acute type B aortic dissection - Thoracic descending aortic dissection - Differences in aortic disease in bicuspid valve patients, regardless of the presence or absence of aortic coarctation. - Angina pectoris, chest pain - Dyspnea accompanied by hypertensive crisis - Sudden coma, or - Shock of unknown cause.

9. The lateral flow device according to any one of claims 1 to 4, wherein the conjugate-binding protein is an FC-binding protein, and preferably the FC-binding protein is protein G, protein A, or protein A / G fusion protein.

10. The lateral flow device according to any one of claims 1 to 4, wherein the detection agent conjugated to the antibody is selected from the group comprising metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules, and preferably the detection agent conjugated to the antibody comprises metallic gold nanoparticles or nanoshells.

11. A lateral flow device according to any one of claims 1 to 10, further comprising a control line positioned substantially perpendicular to the direction of sample flow along a strip, preferably the conjugate pad further comprising immunoglobulin conjugated to a detection agent, wherein during operation the sample flows from the sample pad to the conjugate pad, mobilizing the immunoglobulin conjugate, the immunoglobulin conjugate passes through the detection band without reacting, crosses the control line, preferably the control line is coated with an antibody to which the mobilized immunoglobulin conjugate can bind as it crosses the control line, and optionally the binding at the control line is indicated by a visible color change; more preferably the immunoglobulin in the conjugate is derived from an animal species other than the mammalian species from which the body fluid sample originates.

12. A method for detecting AAD protein in the body fluids of a mammal, comprising using the device described in any one of claims 1 to 11.

13. A method for detecting a first protein (1-AAD-protein) released during an event leading to acute aortic dissection in a mammalian body fluid sample, comprising the following steps: a) The step of placing a sample on a sample pad of a lateral flow assay device, the lateral flow assay device includes the following: a. A strip formed of a material that allows fluid flow by capillary action along a portion of the strip; b. A sample pad positioned near one end of the strip to receive a bodily fluid sample; c. During operation, the sample flows through the strip by capillary action from the sample pad to the conjugate pad, and a first conjugate pad is positioned within the strip to recruit the conjugate contained in the conjugate pad, the conjugate containing a protein antibody that binds to the 1-AAD-protein and is conjugated to a detection agent. d. A first detection band comprising a first conjugate-binding protein immobilized within a strip along a band positioned substantially perpendicular to the direction of sample flow along the strip, wherein when a recruited 1-AAD-protein antibody conjugate in the sample comes into contact with the first conjugate-binding protein in the first detection band, the presence of the 1-AAD-protein in the sample is indicated by a visible color change, wherein the 1-AAD-protein is selected from a group of AAD biomarkers comprising the D-dimer smooth muscle actin myosin isozyme CK-MB and nucleic acid components (DISAMIN), particularly HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, preferably HMGI-C, and the antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1; b) The presence of labeled particles is examined in the detection region to detect whether the AAD protein is present in the sample.

14. A kit for detecting the protein (AAD-protein) released during events leading to acute aortic dissection in mammalian body fluid samples, comprising the following: a) The lateral flow assay device according to any one of claims 1 to 11; and b) Instructions for the use of the device according to any one of claims 1 to 11.

15. A method for determining acute aortic dissection (AAD) in a patient by immunoassay, comprising the following steps: a) Take a sample of the patient's bodily fluids. a. At least one antibody against the first protein (1-AAD-protein) released during the event leading to acute aortic dissection, and b. Incubate with binding partner B for either the I-AAD protein or the antibody. Here, either the antibody or the binding partner B is bound to the detection agent. A step of forming an immunological complex containing a detection agent; b) the step of isolating the immunological complex from the remaining sample; and c) A step of measuring the detection agent in the isolated complex or the remaining sample as an indicator of the 1-AAD- protein in the sample; This determines the occurrence of AAD in a patient, where the 1-AAD-protein is selected from a group of AAD biomarkers including the D-dimer smooth muscle actin myosin isozyme CK-MB and nucleic acid components (DISAMIN), particularly HMGI-C, aggrecan, smooth muscle myosin heavy chain, or soluble aortic elastin, preferably HMGI-C, and the antibody binds to the antigen or a fragment thereof of the amino acid sequence of SEQ ID NO: 1.

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