Use of n-terminal fragments of igfbp-4 in clinical assessment of heart failure, coronary heart disease

Abstract

This invention relates to the field of biomedical technology, specifically to the use of the N-terminal fragment of insulin-like growth factor binding protein-4 (IGF-4) in the clinical assessment of heart failure and coronary artery disease. The N-terminal fragment of IGF-4 is obtained by catalytic hydrolysis of IGF-4 by pregnancy-associated plasma protein A. Clinical assessment of heart failure includes diagnosing or assisting in the diagnosis of heart failure, or assessing or assisting in the prognostic risk of heart failure. Clinical assessment of coronary artery disease includes assessing or assisting in the prognostic risk of coronary artery disease. By detecting the content of the N-terminal fragment of IGF-4 in a sample, heart failure can be diagnosed or assisted in the diagnosis, and the prognosis of heart failure and coronary artery disease can be assessed or assisted in, demonstrating good clinical assessment efficacy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of the N-terminal fragment of IGFBP-4 in the clinical assessment of heart failure and coronary heart disease. Background Technology

[0002] Heart failure (HF) is a complex clinical syndrome caused by abnormal changes in the structure or function of the heart, resulting in an absolute or relative reduction in cardiac output, which fails to meet the needs of the body's tissues and cells. It is the most severe end-stage of cardiovascular disease. With changes in modern lifestyles and the increasing aging of the population, the prevalence of HF is rising continuously, and its high disability and mortality rates have become a significant global public health issue.

[0003] Heart failure presents with diverse clinical manifestations, including dyspnea, fatigue, and edema. Diagnosis relies primarily on medical history, symptoms, signs, and auxiliary examinations, combined with chest X-ray, biomarker testing, and echocardiography to confirm its presence. Echocardiography is the preferred method for assessing cardiac structure and function. Left ventricular ejection fraction (LVEF), measured by echocardiography, shows the amount of blood pumped by the left ventricle with each cardiac contraction. Based on ejection fraction (EF) levels, heart failure can be classified as heart failure with reduced ejection fraction (HFrEF, LVEF < 40%), heart failure with preserved ejection fraction (HFpEF, LVEF ≥ 50%), and heart failure with intermediate ejection fraction (HFmrEF, LVEF 40% to 49%). However, by the time a definitive clinical diagnosis of heart failure is made, the abnormal cardiac structure or function has often already undergone irreversible and profound development. Therefore, there is an urgent need for a clinically applicable method that can assist in the early assessment of heart failure risk. Summary of the Invention

[0004] The purpose of this invention is to provide the use of the N-terminal (hereinafter referred to as N-terminus) fragment of insulin-like growth factor binding protein-4 (hereinafter referred to as IGFBP-4) in the clinical assessment of heart failure and coronary heart disease.

[0005] Therefore, in a first aspect, the present invention provides the use of an antibody that specifically binds to the N-terminal fragment of IGFBP-4 in the preparation of a clinical assessment reagent for heart failure, wherein, The N-terminal fragment of IGFBP-4 is the N-terminal fragment obtained by catalytic hydrolysis of IGFBP-4 by pregnancy-associated plasma protein A (hereinafter referred to as PAPP-A). The heart failure clinical assessment reagent is used to diagnose or assist in the diagnosis of heart failure, or to assess or assist in the assessment of the prognostic risk of heart failure.

[0006] In some embodiments, the N-terminal fragment of the IGFBP-4 includes the amino acid sequence shown in SEQ ID NO: 1.

[0007] In some embodiments, the use further includes: using the antibody that specifically binds to the N-terminal fragment of IGFBP-4 to obtain the detection result of the N-terminal fragment of IGFBP-4 by immunoassay; obtaining the detection result of N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP), and combining the detection result of the antibody on the N-terminal fragment of IGFBP-4, for the diagnosis or auxiliary diagnosis of heart failure, or for the assessment or auxiliary assessment of the prognostic risk of heart failure.

[0008] In some embodiments, the diagnosis or auxiliary diagnosis of heart failure is either (i) or (ii) one of the following: (i) To diagnose or assist in the diagnosis of whether the subject has heart failure; (ii) To diagnose or assist in the diagnosis of the severity of heart failure in the subject.

[0009] In some implementations, the severity refers to left ventricular ejection fraction or NYHA functional class.

[0010] In some implementations, the assessment or auxiliary assessment of the prognostic risk of heart failure is any one of the following (iii) or (iv): (iii) Assess or assist in assessing the short-term prognostic risk of patients with heart failure, where short-term refers to no more than 30 days; (iv) Assess or assist in assessing the long-term prognostic risk of patients with heart failure, where long-term refers to more than 30 days and no more than 240 days.

[0011] In some implementations, the prognostic risk refers to the risk of cardiac readmission and / or death.

[0012] In some embodiments, the heart failure clinical assessment reagent includes a first reagent for detecting the N-terminal fragment of insulin-like growth factor binding protein-4, the first reagent comprising a capture antibody and a detection antibody, wherein one of the capture antibody and the detection antibody is an antibody that specifically binds to NT-IGFBP-4, and the other is an antibody that specifically binds to NT-IGFBP-4 or full-length IGFBP-4.

[0013] In some embodiments, the first reagent is used for a double-antibody sandwich immunoassay (e.g., a double-antibody sandwich chemiluminescent immunoassay).

[0014] In some embodiments, the first reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, fluorescent label, chemiluminescent label, biotin, colloidal gold, electrochemiluminescent label, quantum dot, or enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or microplates.

[0015] In some embodiments, the heart failure clinical assessment reagent further includes a second reagent for detecting N-terminal pro-brain natriuretic peptide, the second reagent being used for immunoassays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay), preferably a double-antibody sandwich immunoassay (e.g., double-antibody sandwich ELISA or CLIA).

[0016] In some embodiments, the second reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, quantum dots, or an enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or a microplate.

[0017] A second aspect of the invention provides the use of an antibody that specifically binds to the N-terminal fragment of IGFBP-4 in the preparation of a clinical assessment reagent for coronary heart disease, wherein, The N-terminal fragment of IGFBP-4 is: the N-terminal fragment obtained by catalytic hydrolysis of IGFBP-4 by PAPP-A; The aforementioned clinical assessment reagent for coronary heart disease is used to assess or assist in assessing the prognostic risk of coronary heart disease.

[0018] In some embodiments, the N-terminal fragment of the IGFBP-4 includes the amino acid sequence shown in SEQ ID NO: 1.

[0019] In some implementations, the coronary prognostic risk refers to the risk of cardiac readmission and / or death.

[0020] In some embodiments, the coronary artery disease clinical assessment reagent includes a first reagent for detecting the N-terminal fragment of insulin-like growth factor binding protein-4, the first reagent comprising a capture antibody and a detection antibody, wherein one of the capture antibody and the detection antibody is an antibody that specifically binds to NT-IGFBP-4, and the other is an antibody that specifically binds to NT-IGFBP-4 or full-length IGFBP-4.

[0021] In some embodiments, the first reagent is used for a double-antibody sandwich immunoassay (e.g., a double-antibody sandwich chemiluminescent immunoassay).

[0022] In some embodiments, the first reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, fluorescent label, chemiluminescent label, biotin, colloidal gold, electrochemiluminescent label, quantum dot, or enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or microplates.

[0023] A third aspect of the present invention provides a method for assessing whether a subject suffers from heart failure or for assessing the prognostic risk of heart failure in a subject, comprising: The content of the N-terminal fragment of IGFBP-4 in samples from subjects was detected, wherein the N-terminal fragment of IGFBP-4 is the N-terminal fragment obtained by catalytic hydrolysis of IGFBP-4 by PAPP-A. The content of the N-terminal fragment of the IGFBP-4 was compared with a reference value, wherein, Compared to the reference value, a higher level of the N-terminal fragment of IGFBP-4 suggests that the subject has heart failure or a higher risk of poor prognosis due to heart failure. Compared to the reference value, a lower level of the N-terminal fragment of IGFBP-4 suggests that the subject does not have heart failure or has a lower risk of poor prognosis due to heart failure.

[0024] In some implementations, a reference value for assessing heart failure is approximately 148.5 ng / mL.

[0025] In some implementations, a reference value for assessing short-term prognostic risk is approximately 314 ng / mL.

[0026] In some implementations, a reference value for assessing long-term prognostic risk is approximately 283 ng / mL.

[0027] In some implementations, detecting the level of the N-terminal fragment of insulin-like growth factor binding protein-4 in a sample from a subject includes the following steps: Provide samples from the subjects; A reagent is provided, the reagent comprising an antibody that specifically binds to the N-terminal fragment of insulin-like growth factor binding protein-4; using the reagent, the sample is detected by an immunoassay to obtain the detection result of the content of the N-terminal fragment of insulin-like growth factor binding protein-4.

[0028] In some implementations, detecting the level of the N-terminal fragment of insulin-like growth factor binding protein-4 in a sample from a subject includes the following steps: The reagents provided include: a magnetic solid-phase support coated with a capture antibody, and an alkaline phosphatase-labeled detection antibody; wherein at least one of the capture antibody and the detection antibody can specifically bind to the N-terminal fragment of IGFBP-4; Provide samples from the subjects; The sample is mixed with the magnetic solid-phase carrier coated with the capture antibody and the alkaline phosphatase-labeled detection antibody, and incubated to form an immune complex; Provide magnetic field conditions to clean the immune complex and remove unbound substances; A chemiluminescent substrate, suitable for being decomposed by alkaline phosphatase and producing chemiluminescence, is added to the washed immune complex; the intensity of the chemiluminescence is detected, and the content of the N-terminal fragment of IGFBP-4 is calculated.

[0029] In some embodiments, the method for assessing whether a subject has heart failure or assessing the risk of heart failure prognosis in a subject further includes: The levels of NT-proBNP in samples from subjects are detected; based on the N-terminal fragment of IGFBP-4 and the levels of NT-proBNP, it is determined whether the subject has heart failure or to assess the subject's prognostic risk of heart failure.

[0030] In some embodiments, the content of the N-terminal fragment of IGFBP-4 and the content of NT-proBNP are compared with their respective reference values, wherein, When the content of the N-terminal fragment of IGFBP-4 and the content of NT-proBNP are both higher than their respective reference values, it indicates that the subject has heart failure or has a high risk of poor prognosis due to heart failure. When the levels of the N-terminal fragment of IGFBP-4 and the NT-proBNP are both lower than their respective reference values, it indicates that the subject has no heart failure or has a low risk of poor prognosis due to heart failure.

[0031] In some implementations, the reference value for NT-proBNP used to assess whether heart failure is present is approximately 125 ng / L, and the reference value for NT-IGFBP-4 is approximately 220 ng / mL.

[0032] In some implementations, the reference value for NT-proBNP used to assess short-term prognosis is approximately 3110 ng / L, and the reference value for NT-IGFBP-4 is approximately 314 ng / mL.

[0033] In some implementations, the reference value for NT-proBNP used to assess long-term prognosis is approximately 3110 ng / L, and the reference value for NT-IGFBP-4 is approximately 283 ng / mL.

[0034] A fourth aspect of the present invention provides a method for assessing the prognostic risk of coronary heart disease, comprising: The content of the N-terminal fragment of IGFBP-4 in samples from patients with coronary heart disease was detected, wherein the N-terminal fragment of IGFBP-4 is the N-terminal fragment obtained by catalytic hydrolysis of IGFBP-4 by PAPP-A. The content of the N-terminal fragment of the IGFBP-4 was compared with a reference value, wherein, Compared to the reference value, higher levels of the N-terminal fragment of IGFBP-4 suggest a higher risk of poor prognosis for coronary heart disease in the subject. Compared to the reference value, a lower level of the N-terminal fragment of IGFBP-4 suggests that the subject has a lower risk of poor prognosis in coronary artery disease.

[0035] In some implementations, the reference value for assessing the prognosis of coronary artery disease is approximately 174 ng / mL.

[0036] In some embodiments, the N-terminal fragment of the IGFBP-4 includes the amino acid sequence shown in SEQ ID NO: 1.

[0037] A fifth aspect of the present invention provides a kit comprising a first reagent for detecting an N-terminal fragment of insulin-like growth factor binding protein-4 and a second reagent for detecting an N-terminal precursor of brain natriuretic peptide.

[0038] In some embodiments, the first reagent comprises a capture antibody and a detection antibody, wherein one of the capture antibody and the detection antibody is an antibody that specifically binds to NT-IGFBP-4, and the other is an antibody that specifically binds to NT-IGFBP-4 or full-length IGFBP-4.

[0039] In some embodiments, the first reagent is used for a double-antibody sandwich immunoassay (e.g., a double-antibody sandwich chemiluminescent immunoassay).

[0040] In some embodiments, the first reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, fluorescent label, chemiluminescent label, biotin, colloidal gold, electrochemiluminescent label, quantum dot, or enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or microplates.

[0041] In some embodiments, the second reagent is used for immunoassays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay), preferably double-antibody sandwich immunoassays (e.g., double-antibody sandwich ELISA or CLIA).

[0042] In some embodiments, the second reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, quantum dots, or an enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or a microplate.

[0043] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other objects, features and advantages of the present invention more obvious and understandable, the following lists specific embodiments of the present invention. Attached Figure Description

[0044] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. In the drawings: Figure 1 Flowchart for the sample inclusion of healthy individuals and those with heart failure; Figure 2 The concentration of NT-IGFBP-4 in healthy individuals and those with heart failure; Figure 3 : Heart failure diagnostic performance analysis process of the NT-IGFBP-4 kit; Figure 4 ROC curve of NT-IGFBP-4 for heart failure diagnosis; Figure 5 Diagnostic decision tree using combined NT-IGFBP-4 and NT-proBNP Figure 6 Correlation analysis results between NT-IGFBP-4 concentration levels and left ventricular ejection fraction (LVEF%); Figure 7 The results of the correlation analysis between NT-IGFBP-4 concentration levels and NYHA functional class scores; Figure 8 Flowchart of sample inclusion and exclusion for heart failure prognosis studies; Figure 9Kaplan-Meier survival curves during a 240-day follow-up period for heart failure patients when NT-IGFBP-4 was used alone to assess the prognostic risk of heart failure; the upper curve represents the low-risk group and the lower curve represents the high-risk group. Figure 10 Kaplan-Meier survival curves during a 240-day follow-up period for heart failure patients when NT-IGFBP-4 and NT-proBNP were used in combination to assess the prognostic risk of heart failure; the three curves, from top to bottom, represent the low-risk group, intermediate-risk group, and high-risk group. Figure 11 Kaplan-Meier survival curves during a 30-day follow-up period for heart failure patients when NT-IGFBP-4 is used alone to assess the prognostic risk of heart failure; the upper curve represents the low-risk group and the lower curve represents the high-risk group. Figure 12 Kaplan-Meier survival curves during a 30-day follow-up period for heart failure patients when NT-proBNP is used alone to assess the prognostic risk of heart failure; the upper curve represents the low-risk group and the lower curve represents the high-risk group. Figure 13 Kaplan-Meier survival curves during a 30-day follow-up period in the heart failure population when NT-IGFBP-4 and NT-proBNP were used in combination to assess the prognostic risk of heart failure; the three curves, from top to bottom, represent the low-risk group, the intermediate-risk group, and the high-risk group. Figure 14 Flowchart of sample inclusion and exclusion for coronary artery disease prognosis studies; Figure 15 Kaplan-Meier survival curves during a 120-day follow-up period for patients with coronary artery disease when using NT-IGFBP-4 to assess the prognostic risk of coronary artery disease; the upper curve represents the low-risk group and the lower curve represents the high-risk group. Detailed Implementation

[0045] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0046] the term As used herein, the terms "N-terminal fragment of insulin-like growth factor binding protein-4," "N-terminal fragment of IGFBP-4," and "NT-IGFBP-4" have the same meaning. IGFBP-4 is a member of the insulin-like growth factor binding protein (IGFBP) superfamily. The human IGFBP-4 gene is located on human chromosome 17P13-12, is 15.3 kb in length, and consists of 4 exons and 3 introns. Human IGFBP-4 (see amino acid residues 22-258 of the protein corresponding to UniProt database accession number P22692) consists of 237 amino acids, and the original product contains a 21-amino acid signal peptide. Under physiological conditions in vivo, IGFBP-4 can be cleaved at specific sites by pregnancy-associated plasma protein-A (PAPP-A), releasing the N-terminal and C-terminal fragments of IGFBP-4 into the bloodstream. The N-terminal fragment of human IGFBP-4 has the amino acid sequence shown in SEQ ID NO: 1.

[0047] SEQ ID NO: 1 DEAIHCPPCSEEKLARCRPPVGCEELVREPGCGCCATCALGLGMPCGVYTPRCGSGLRCYPPRGVEKPLHTLMHGQGVCMELAEIEAIQESLQPSDKDEGDHPNNSFSPCSAHDRRCLQKHFAKIRDRSTSGGKM As used in this article, the terms "heart failure" and "heart failure" have the same meaning: a clinical syndrome caused by various structural or functional diseases of the heart, resulting in impaired ventricular filling and ejection function, which in turn prevents the cardiac output from meeting the metabolic needs of the body's tissues. This condition is mainly characterized by symptoms such as dyspnea, limited physical activity, and fluid retention, and is characterized by high mortality and poor prognosis.

[0048] As used in this article, the terms "N-terminal pro-BNP," "N-terminal pro-BNP," and "NT-proBNP" have the same meaning: a linear monopeptide secreted by the ventricles. When cardiomyocytes are stimulated, they produce a 134-amino acid precursor to B-type natriuretic peptide (pre-proBNP), which then forms a 108-amino acid precursor to BNP (proBNP). The latter is further cleaved by endonucleases into NT-proBNP and BNP. Due to its long half-life and relative stability in vivo, NT-proBNP has become a diagnostic biomarker recognized by clinical guidelines for heart failure. Methods for detecting NT-proBNP are well known to those skilled in the art, and commercially available kits are available, such as: NT-proBNP Detection Kit (Mindray, catalog number #105-027041-00), Human NT-proBNP ELISA Kit (Abcam, catalog number #ab263877), Human proBNP / NPPB ELISA Kit (Invitrogen, catalog number #EHPRONPPB), and LumiraDx NT-proBNP Detection Kit (Roche, catalog number #10418320001).

[0049] As used herein, the term "specifically binds to the N-terminal fragment of IGFBP-4" refers to an antibody or its antigen-binding fragment that recognizes and binds to the N-terminal fragment of IGFBP-4 and exhibits minimal cross-reactivity with other proteins, such as full-length IGFBP-4 and the C-terminal fragment of IGFBP-4. In some embodiments, the recognition epitope of the antibody that specifically binds to NT-IGFBP-4 includes a novel epitope formed at the end of the N-terminus of IGFBP-4 after full-length IGFBP-4 is cleaved by pregnancy-associated plasma protein-A (PAPP-A), which is not present in full-length IGFBP-4. For example, in some embodiments, the antibody or its antigen-binding fragment that specifically binds to the N-terminal fragment of IGFBP-4 exhibits less than 5% cross-reactivity with full-length IGFBP-4.

[0050] As used in this article, the term "coronary heart disease" refers to coronary atherosclerotic heart disease, a heart disease caused by lesions such as atherosclerosis, thromboembolism, or vasospasm in the coronary arteries, leading to narrowing or even blockage of the lumen, which in turn causes myocardial ischemia, hypoxia, or necrosis.

[0051] As used herein, the terms “patient” and “subject” refer to an organism, such as a mammal, and in some embodiments, the subject is a human.

[0052] As used herein, the term "sample" refers to a substance obtained from a subject or isolated tissue, cells, or body fluid that is suitable for the detection of a biomarker (e.g., NT-IGFBP-4). In some embodiments, a sample may be blood, serum, plasma, cerebrospinal fluid, lymph, spinal fluid, mucosal secretions, exudate, amniotic fluid, synovial fluid, peritoneal fluid, etc.

[0053] As used herein, the term "ROC curve" refers to the Receiver Operating Characteristic curve. In certain specific embodiments of the invention, the ROC curve refers to the ROC curve between the true positive rate and the false positive rate. The area under the ROC curve is the AUC, and ROC curves and AUC are commonly used to evaluate the efficacy of clinical assessments.

[0054] As used in this article, the terms “high expression” and “above reference value” have the same meaning, referring to an increase of at least 5%, 10%, 20%, 30%, 50%, 80%, 100% or more compared to the “reference value” or “threshold”.

[0055] As used in this invention, the terms “low expression” and “below reference value” have the same meaning, referring to a reduction of at least 5%, 10%, 20%, 30%, 50%, 80%, or more significant compared to a “reference value” or “threshold”.

[0056] As used in this invention, the setting of "reference values" or "thresholds" for gene or protein expression is readily achievable by those skilled in the art based on the spirit of this invention. Selecting appropriate "reference values" or "thresholds" is a routine part of experimental design; for example, a statistically significant analysis can first be performed on the corresponding protein expression levels in samples from subjects (patients) with confirmed diagnoses or well-defined prognoses, and the obtained expression values ​​can be used as "reference values" or "thresholds." Alternatively, in some embodiments, thresholds recommended by clinical guidelines can be used for certain biomarkers such as NT-proBNP.

[0057] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, "antibody" includes not only complete (i.e., full-length) antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv, scFv, Fd, CDR fragments), their variants, fusion proteins containing antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of an immunoglobulin molecule containing a desired specific antigen recognition site, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. Antibodies can be derived from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, rats, mice, etc., or other animals such as birds (e.g., chickens), fish (e.g., sharks), and camels (e.g., llamas).

[0058] Typically, a complete or full-length antibody consists of two heavy chains and two light chains. Each heavy chain contains a variable region (VH) and a constant region (CH); each light chain contains a variable region (VL) and a constant region (CL). Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses of these classes), but antibodies do not necessarily belong to any specific class. Immunoglobulins can be classified into different classes based on the amino acid sequence of their antibody heavy chain constant regions. Generally, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0059] As used herein, the terms "antigen-binding fragment" or "antigen-binding moiety" refer to the portion or region of an intact antibody molecule responsible for binding to an antigen. The antigen-binding moiety may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. VH and VL typically each contain three complementarity-determining regions (CDR1, CDR2, and CDR3). Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, bispecific antibodies, single-domain antibodies, chimeric antibodies, linear antibodies, nanobodies (from Domantis's technology), proantibodies, and such peptides containing at least a portion of the antibody sufficient to confer specific antigen-binding ability to the peptide.

[0060] As used herein, the term "immunoassay" refers to a method that utilizes the specific interaction / binding affinity between an antigen and an antibody, typically used to detect the presence or level of a specific antigen or antibody in a sample. Such immunoassays are well known to those skilled in the art and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, immunoturbidimetry, and surface plasmon resonance assays.

[0061] As used herein, the term "double antibody sandwich assay" specifically refers to a commonly used immunoassay technique in which one antigen binds to two different antibodies. The antibodies bind to two different, non-overlapping, and non-interfering epitopes of the antigen. In this assay, a sandwich structure is formed comprising a capture antibody, an antigen, and a detection antibody, thereby bridging the antigen to the two antibodies bound to it. In some embodiments, the detection antibody carries a detection label, such as a radiolabel, fluorescent label, chemiluminescent label, biotin, colloidal gold, electrochemiluminescent label, quantum dot, or enzyme. In some embodiments, the detection label is selected from enzymes, such as alkaline phosphatase. In some embodiments, the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or a microplate. In some embodiments, the capture antibody is coated on the surface of magnetic beads.

[0062] As used herein, the term “about” means ±10% of the range or value (e.g., ±5%, ±2% or ±1%), unless otherwise stated.

[0063] Products and methods for diagnosing or assisting in the diagnosis of heart failure In some embodiments, the use of antibodies that specifically bind to the N-terminal fragment of IGFBP-4 is provided in the preparation of reagents for the diagnosis or auxiliary diagnosis of heart failure.

[0064] In some embodiments, antibodies that specifically bind to the N-terminal fragment of IGFBP-4 are used alone to prepare the reagents for diagnosing or assisting in the diagnosis of heart failure. In some embodiments, antibodies that specifically bind to the N-terminal fragment of IGFBP-4 are combined with reagents for detecting NT-proBNP to prepare the reagents for diagnosing or assisting in the diagnosis of heart failure.

[0065] In some embodiments, the use includes: using the antibody that specifically binds to the N-terminal fragment of insulin-like growth factor binding protein-4 to obtain the detection result of the N-terminal fragment of insulin-like growth factor binding protein-4 by immunoassay; obtaining the detection result of the N-terminal brain natriuretic peptide precursor, and combining the detection result of the antibody of the N-terminal fragment of insulin-like growth factor binding protein-4 for the diagnosis or auxiliary diagnosis of heart failure.

[0066] In some embodiments, the diagnosis or auxiliary diagnosis of heart failure is either (i) or (ii) one of the following: (i) To diagnose or assist in the diagnosis of whether the subject has heart failure; (ii) To diagnose or assist in the diagnosis of the severity of heart failure in the subject.

[0067] In some implementations, the severity refers to left ventricular ejection fraction (LVEF%) or NYHA functional class. LVEF, the percentage of stroke volume to end-diastolic ventricular volume, reflects left ventricular systolic function and is a crucial indicator for clinically assessing the severity of heart failure. The NYHA functional class, developed by the New York Heart Association, is divided into four levels (I-IV) and is a common clinical method for assessing the degree of cardiac function impairment, frequently used to reflect the severity of heart failure. NYHA class I is the mildest, indicating that the patient has heart disease but is not limited in physical activity; NYHA class IV is the most severe, indicating that the patient has heart disease, experiences heart failure or angina symptoms at rest, and discomfort increases with any physical activity.

[0068] In some implementations, a method for assessing whether a subject suffers from heart failure is provided, comprising: The levels of the N-terminal fragment of IGFBP-4 in samples from subjects were detected; The content of the N-terminal fragment of the IGFBP-4 was compared with a reference value, wherein, A higher level of the N-terminal fragment of IGFBP-4 compared to the reference value suggests that the subject has heart failure. The lower levels of the N-terminal fragment of IGFBP-4 compared to the reference value suggest that the subject does not have heart failure.

[0069] In some implementations, the method for assessing whether a subject suffers from heart failure includes the following steps: The reagent provided comprises: a magnetic solid-phase support coated with a capture antibody, and an alkaline phosphatase-labeled detection antibody; wherein at least one of the capture antibody and the detection antibody specifically binds to the N-terminal fragment of IGFBP-4; Provide samples from the subjects; The sample was mixed with the magnetic solid-phase carrier coated with the capture antibody and the alkaline phosphatase-labeled detection antibody, and incubated to obtain an immune complex. Provide magnetic field conditions to clean the immune complex and remove unbound substances; A chemiluminescent substrate, suitable for being decomposed by alkaline phosphatase and producing chemiluminescence, is added to the washed immune complex; the intensity of the chemiluminescence is detected, and the content of the N-terminal fragment of IGFBP-4 is calculated.

[0070] In some embodiments, the method for assessing whether a subject suffers from heart failure further includes: The levels of NT-proBNP in samples from subjects are detected; based on the N-terminal fragment of IGFBP-4 and the levels of NT-proBNP, it is determined whether the subject has heart failure or to assess the subject's prognostic risk of heart failure.

[0071] In some embodiments, the content of the N-terminal fragment of IGFBP-4 and the content of NT-proBNP are compared with their respective reference values, wherein, When the NT-proBNP level is lower than its reference value, it indicates that the subject does not have heart failure; If the levels of the N-terminal fragment of IGFBP-4 and the NT-proBNP are both higher than their respective reference values, it indicates that the subject has heart failure.

[0072] Products and methods for assessing or assisting in the assessment of the prognostic risk of heart failure In some embodiments, the use of antibodies that specifically bind to the N-terminal fragment of IGFBP-4 is provided in the preparation of reagents for assessing or assisting in the assessment of the prognostic risk of heart failure.

[0073] In some embodiments, antibodies that specifically bind to the N-terminal fragment of IGFBP-4 are used alone to prepare the product for assessing or assisting in the assessment of the prognostic risk of heart failure. In some embodiments, antibodies that specifically bind to the N-terminal fragment of IGFBP-4 are combined with reagents for detecting NT-proBNP to prepare the reagent for assessing or assisting in the assessment of the prognostic risk of heart failure.

[0074] In some embodiments, the use includes: using the antibody that specifically binds to the N-terminal fragment of insulin-like growth factor binding protein-4 to obtain the detection result of the N-terminal fragment of insulin-like growth factor binding protein-4 by immunoassay; obtaining the detection result of the N-terminal brain natriuretic peptide precursor, and combining the detection result of the antibody of the N-terminal fragment of insulin-like growth factor binding protein-4, for assessing or assisting in the assessment of the prognostic risk of heart failure.

[0075] In some implementations, the assessment or auxiliary assessment of the prognostic risk of heart failure is any one of the following (iii) or (iv): (iii) Assess or assist in assessing the short-term prognostic risk of patients with heart failure, where short-term refers to no more than 30 days; (iv) Assess or assist in assessing the long-term prognostic risk of patients with heart failure, where long-term refers to more than 30 days and no more than 240 days.

[0076] In some implementations, the prognostic risk refers to the risk of cardiac readmission and / or death.

[0077] In some implementations, a method for assessing the prognostic risk of heart failure in a subject is provided, comprising: The levels of the N-terminal fragment of IGFBP-4 in samples from subjects were detected; The content of the N-terminal fragment of the IGFBP-4 was compared with a reference value, wherein, Compared to the reference value, higher levels of the N-terminal fragment of IGFBP-4 suggest a higher risk of poor prognosis in the subject with heart failure. Compared to the reference value, lower levels of the N-terminal fragment of IGFBP-4 suggest a lower risk of poor prognosis in the subject with heart failure.

[0078] In some implementations, the method for assessing the prognostic risk of heart failure in a subject includes the following steps: The reagent provided includes: a magnetic solid-phase support coated with a capture antibody, and an alkaline phosphatase-labeled detection antibody; wherein at least one of the capture antibody and the detection antibody can specifically bind to the N-terminal fragment of IGFBP-4; Provide samples from the subjects; The sample was mixed with the magnetic solid-phase carrier coated with the capture antibody and the alkaline phosphatase-labeled detection antibody, and incubated to obtain an immune complex. Provide magnetic field conditions to clean the immune complex and remove unbound substances; A chemiluminescent substrate, suitable for being decomposed by alkaline phosphatase and producing chemiluminescence, is added to the washed immune complex; the intensity of the chemiluminescence is detected, and the content of the N-terminal fragment of IGFBP-4 is calculated.

[0079] In some embodiments, the method for assessing the prognostic risk of heart failure in a subject further includes: The levels of NT-proBNP in samples from subjects are detected; based on the N-terminal fragment of IGFBP-4 and the levels of NT-proBNP, it is determined whether the subject has heart failure or to assess the subject's prognostic risk of heart failure.

[0080] In some embodiments, the content of the N-terminal fragment of IGFBP-4 and the content of NT-proBNP are compared with their respective reference values, wherein, When the levels of the N-terminal fragment of IGFBP-4 and the NT-proBNP are both higher than their respective reference values, it indicates that the subject has a higher risk of poor prognosis in heart failure. If the content of the N-terminal fragment of IGFBP-4 or the content of NT-proBNP is higher than their respective reference values, it indicates that the subject has a moderate risk of poor prognosis of heart failure. When the levels of the N-terminal fragment of IGFBP-4 and the NT-proBNP are both lower than their respective reference values, it indicates that the subject has a lower prognostic risk of heart failure.

[0081] Products and methods for assessing or assisting in the assessment of the prognostic risk of coronary artery disease In some embodiments, the use of antibodies that specifically bind to the N-terminal fragment of IGFBP-4 is provided in the preparation of reagents for assessing or assisting in the assessment of the prognostic risk of coronary artery disease.

[0082] In some implementations, the coronary prognostic risk refers to the risk of cardiac readmission and / or death.

[0083] In some embodiments, a method for assessing the prognostic risk of coronary artery disease is provided, comprising: The content of the N-terminal fragment of IGFBP-4 was detected in samples from patients with coronary artery disease; The content of the N-terminal fragment of the IGFBP-4 was compared with a reference value, wherein, Compared to the reference value, higher levels of the N-terminal fragment of IGFBP-4 suggest a higher risk of poor prognosis for coronary heart disease in the subject. Compared to the reference value, a lower level of the N-terminal fragment of IGFBP-4 suggests that the subject has a lower risk of poor prognosis in coronary artery disease.

[0084] Reagent test kit In some embodiments, a kit for detecting NT-IGFBP-4 is provided, which allows for the detection of NT-IGFBP-4 by a double-antibody sandwich chemiluminescent immunoassay.

[0085] The kit includes: The working solution of the magnetic bead coating includes a magnetic solid-phase carrier (e.g., a mixture of superparamagnetic microparticles) coated with the capture antibody.

[0086] Enzyme-labeled working solutions, including alkaline phosphatase-labeled detection antibodies.

[0087] At least one of the capture antibody and the detection antibody is an antibody that specifically binds to NT-IGFBP-4, and the capture antibody, the detection antibody, and NT-IGFBP-4 can form an immune complex. In some embodiments, the recognition epitope of the antibody that specifically binds to NT-IGFBP-4 includes a new epitope formed at the end of the N-segment of IGFBP-4 after full-length IGFBP-4 is cleaved by pregnancy-associated plasma protein-A (PAPP-A). Since this new epitope structure is not present in full-length IGFBP-4, it cannot be recognized by the antibody.

[0088] In some embodiments, one of the capture antibody and the detection antibody is an antibody that specifically binds to NT-IGFBP-4, and the other can recognize and bind to NT-IGFBP-4 or full-length IGFBP-4.

[0089] The aforementioned capture and detection antibodies can be obtained commercially, such as the monoclonal antibody Cat.#4IGF4 purchased from Hytest, as detailed below: The monoclonal antibody IBP3cc (4IGF4-IBP3cc) specifically binds to human NT-IGFBP-4, and its binding rate to full-length human IGFBP-4 is <5% (ELISA); other antibodies that can specifically bind to the new epitope structure formed at the N-terminus of IGFBP-4 after PAPP-A cleavage are also applicable to this application.

[0090] Monoclonal antibodies IBP144, IBP154, and IBP180 (4IGF4-IBP144, 4IGF4-IBP154, 4IGF4-IBP180) can bind to human IGFBP-4 and human NT-IGFBP-4. The epitopes of these antibodies are located in the amino acid sequence 1-156 of IGFBP-4. Other antibodies that can recognize the epitopes located in the amino acid sequence 1-156 of IGFBP-4 are also applicable to this application.

[0091] In some embodiments, the capture antibody may be monoclonal antibody IBP3cc, and the detection antibody may be selected from monoclonal antibodies IBP144, IBP154, and IBP180. In other embodiments, the capture antibody may be selected from monoclonal antibodies IBP144, IBP154, and IBP180, and the detection antibody may be monoclonal antibody IBP3cc. Preferably, the capture antibody is an antibody that specifically binds to human NT-IGFBP-4.

[0092] In some embodiments, when using a sandwich immunoassay to detect human NT-IGFBP-4, the combination of capture antibody and detection antibody may be selected from any of the following groups: IBP3cc-IBP144; IBP3cc-IBP180; IBP3cc-IBP154.

[0093] The above kit can be used with Mindray's fully automated chemiluminescence analyzers CL2000i, CL6000i, and CL8000i, and the principle and process for NT-IGFBP-4 detection are as follows: Step 1: Add the sample, magnetic bead working solution, and enzyme-labeled working solution to the reaction tube. After incubation, NT-IGFBP-4 in the sample binds to the capture antibody coated on the magnetic beads. Simultaneously, the binding of the antibody-alkaline phosphatase label to NT-IGFBP-4 in the sample is detected. After the reaction is complete, the solid phase is placed in a magnetic field. The magnetic field attracts the magnetic beads, and the substances bound to the solid phase are retained, while unbound substances are washed away.

[0094] Step 2: The chemiluminescent substrate is added to the reaction tube. The substrate (3-(2-spirodaradinane)-4-methoxy-4-(3-phosphoyl)-phenyl-1,2-dioxane, AMPPD) is decomposed by alkaline phosphatase, losing a phosphate group to generate an unstable intermediate. This intermediate generates a methyl m-oxobenzoate anion through intramolecular electron transfer. When the excited-state methyl m-oxobenzoate anion returns to the ground state, chemiluminescence is produced. The number of photons generated in the reaction is then measured using a photomultiplier tube. The number of photons generated is proportional to the concentration of NT-IGFBP-4 in the sample. The amount of analyte in the sample is determined by a calibration curve.

[0095] The following description provides examples of the invention, thereby making the advantages and various effects of the invention more clearly apparent. Those skilled in the art will understand that these examples are illustrative and not intended to limit the invention.

[0096] Example 1 A kit for detecting NT-IGFBP-4 is provided. Using this kit, NT-IGFBP-4 can be detected by a double-antibody sandwich chemiluminescent immunoassay.

[0097] The kit includes: The magnetic bead coating working solution comprises a mixture of superparamagnetic microparticles coated with capture antibodies. This working solution is used to achieve the capture of NT-IGFBP-4 in samples.

[0098] The enzyme-labeled working solution includes an alkaline phosphatase-labeled detection antibody. This solution is used to detect the NT-IGFBP-4 antigen trapped by superparamagnetic microparticles.

[0099] The capture antibody was monoclonal antibody IBP3cc, and the detection antibody was monoclonal antibody IBP144.

[0100] The above kit was used in conjunction with the Mindray CL8000i fully automated chemiluminescence analyzer for the detection of NT-IGFBP-4 in subsequent examples.

[0101] Example 2 This embodiment analyzes the differences in NT-IGFBP-4 levels between apparent healthy individuals and those with heart failure.

[0102] To evaluate the application of the NT-IGFBP-4 assay kit under routine clinical conditions, the concentration levels of NT-IGFBP-4 in plasma samples from patients diagnosed with heart failure (n=35) and apparent healthy individuals (n=62) were investigated. The apparent healthy population was further screened from self-reported healthy adults undergoing physical examinations based on exclusion criteria, including eGFR>60, HbA1c (glycated hemoglobin) <6.5% (or <48 mmol / mL), NT-proBNP <125 pg / mL, and hs-TnI <31.3 ng / L (male) or 15.3 ng / L (female). The heart failure patients were diagnosed by at least one cardiologist based on their medical history, symptoms, signs, and auxiliary examinations such as echocardiography. The inclusion process for both groups is described below. Figure 1 Blood samples were collected from both groups upon admission using lithium heparin vacuum blood collection tubes (BD, CNL20-C0371). After processing, the samples were tested using the NT-IGFBP-4 kit provided in Example 1. Statistical analysis was performed using Graph PadPrism 9.0 software. The t-test (normal distribution) was used for comparisons of continuous data between groups. A statistically significant difference was considered to be P < 0.05.

[0103] The results are as follows Figure 2 As shown, the concentration of NT-IGFBP-4 differed significantly between the apparent healthy population and the heart failure population (statistical difference P<0.0001). The mean NT-IGFBP-4 concentration in the healthy population was 89.53, while the mean in the heart failure population significantly increased to 181.11. A significant correlation was found between NT-IGFBP-4 protein concentration and heart failure, suggesting that NT-IGFBP-4 may have important application value in the diagnosis and prognosis of heart failure.

[0104] Example 3 This embodiment is used to evaluate the application value of NT-IGFBP-4 in the diagnosis of heart failure.

[0105] This study consecutively included 189 patients who presented with suspected heart failure symptoms such as dyspnea, chest tightness, wheezing, shortness of breath, and shortness of breath at the hospital emergency department. Two cardiologists made the final clinical diagnosis based on the patients' medical records, including chief complaints, medical history, physical examination, chest X-ray, electrocardiogram, and echocardiography. Of these, 70 patients were diagnosed with heart failure according to clinical criteria, and 119 patients were diagnosed with heart failure according to clinical criteria. For detailed sample inclusion procedures, please refer to [link to sample inclusion process]. Figure 3 .

[0106] Blood samples were collected from patients upon their arrival at the hospital on the same day. Sampling was performed using lithium heparin vacuum blood collection tubes (BD, CNL20-C0371). After processing, the samples were tested using the NT-IGFBP-4 kit provided in Example 1. Statistical analysis was performed using IBM SPSS Statistics (27.0), calculating the area under the receiver operating characteristic curve (ROC), sensitivity, and specificity. A statistically significant difference was considered to be p < 0.05. Results are as follows: Figure 4 As shown in Table 1, the area under the ROC curve (AUC) of NT-IGFBP-4 in the diagnosis of heart failure was 0.841. When the Youden index was at its maximum of 0.564, the optimal diagnostic threshold was 148.5 ng / mL, at which point the sensitivity and specificity were 75.7% and 80.7%, respectively. The negative predictive value (85.0%) was more significant than the positive predictive value (69.7%).

[0107] Table 1

[0108] Example 4 This embodiment is used to evaluate the application value of the combined use of NT-IGFBP-4 and NT-proBNP in the diagnosis of heart failure.

[0109] Based on Example 3, NT-proBNP was detected in samples from 189 patients (Mindray, #105-027041-00), and the diagnostic value of NT-proBNP alone, NT-IGFBP-4 alone, and the combined use of NT-IGFBP-4 and NT-proBNP in assessing heart failure was analyzed. Specifically, in the case of combined diagnosis using NT-IGFBP-4 and NT-proBNP, based on... Figure 5 The joint diagnostic decision tree is used to determine the positive or negative nature of heart failure.

[0110] Patients were divided into NT-proBNP negative and NT-proBNP positive groups based on the NT-proBNP guideline threshold, with the NT-proBNP negative group corresponding to the heart failure negative group. Within the NT-proBNP positive group, the diagnostic threshold of NT-IGFBP-4 was also used for further subdivision, with the NT-IGFBP-4 positive group corresponding to the heart failure positive group and the NT-IGFBP-4 negative group serving as the observation group, requiring confirmation based on other clinical indicators such as echocardiography. The diagnostic threshold for NT-IGFBP-4 (220 ng / mL) was calculated based on a specificity of at least 85%. The efficacy results of the combined application of the NT-IGFBP-4 and NT-proBNP diagnostic decision tree are shown in Table 2. Figure 5 As shown.

[0111] Table 2

[0112] See Table 2 and Figure 5 When NT-IGFBP-4 and NT-proBNP are used together for diagnosis, the specificity is significantly improved from 67.2% to 88% while maintaining high sensitivity, and the positive predictive value (PPV) is increased from 63.6% to 82.4%. This indicates that when the clinical NT-proBNP level is below the diagnostic threshold, there is a 97.6% probability that the patient is negative for heart failure, and the diagnosis can be safely ruled out; when both levels are above the diagnostic threshold, there is an 82.4% probability that the patient is positive for heart failure, and clinical treatment should be considered; when NT-proBNP is positive and NT-IGFBP-4 is negative, other clinical methods should be used to observe the patient.

[0113] That is, in this embodiment, the sensitivity of NT-proBNP in excluding heart failure can reach 97.1%, but its specificity is low when used alone, only 67.2%. However, by combining it with NT-IGFBP-4 for diagnosis, the diagnostic specificity and positive predictive value (PPV) can be significantly improved.

[0114] NT-proBNP, a widely used clinical marker for heart failure, is highly sensitive and valuable in ruling out heart failure with negative results. However, due to its extremely low specificity (only 67.2% in this example) and PPV, NT-proBNP-positive patients often require confirmation using multiple other clinical tests such as echocardiography and electrocardiography. Combining NT-IGFBP-4 with NT-proBNP significantly improves diagnostic specificity and PPV. This allows approximately one-third of NT-proBNP-positive patients to be considered for treatment without further testing, saving medical costs, avoiding resource consumption, and improving hospital turnover.

[0115] In summary, by using NT-IGFBP-4 and NT-proBNP in combination, clinically diagnosed patients can be divided into three categories: those with negative heart failure results can be safely excluded; those with positive heart failure results can be considered for treatment; and those under observation require further examination for final confirmation. Compared to using NT-proBNP alone, this combined approach significantly improves the specificity and PPV of heart failure diagnosis while maintaining high sensitivity and NPV, bringing good benefits to clinical patients.

[0116] Example 5 Left ventricular ejection fraction (LVEF) and the NYHA functional class score are both indicators for evaluating the severity of heart failure. This embodiment evaluates the correlation between NT-IGFBP-4 and LVEF and the NYHA functional class score. According to the evaluation results, the measured value of NT-IGFBP-4 can be used to diagnose or assist in the diagnosis of the severity of heart failure in the subjects.

[0117] I. Left ventricular ejection fraction (LVEF) LVEF (Left Ventricular Expectorant Emission) refers to the percentage of stroke volume delivered to the end-diastolic volume of the ventricle. It reflects left ventricular systolic function and is an important indicator for clinically assessing the severity of heart failure. Generally, a normal LVEF value should be ≥50%, while a value <50% should raise suspicion of heart failure.

[0118] A total of 136 cardiology patients with LVEF measurements during their outpatient or emergency visit were consecutively randomly enrolled. Blood samples were collected from patients on the day of their visit using lithium heparin vacuum blood collection tubes (BD, CNL20-C0371). After processing, the samples were tested using the NT-IGFBP-4 kit provided in Example 1. Statistical analysis was performed using GraphPad Prism 9.0, and a two-sided significance level of P < 0.05 was considered statistically significant.

[0119] like Figure 6 As shown, NT-IGFBP-4 concentration levels were significantly correlated with left ventricular ejection function (P < 0.001). Higher NT-IGFBP-4 concentrations were associated with lower left ventricular ejection fraction (LVEF%), indicating more severe heart failure. These findings demonstrate that NT-IGFBP-4 concentration levels are significantly correlated with left ventricular systolic function, and its measurement can reflect the severity of heart failure in the subjects.

[0120] II. NYHA Cardiac Function Class The NYHA functional class, developed by the New York Heart Association, is divided into four levels, I-IV. It is a common clinical method for assessing the degree of impairment of cardiac function and is often used to reflect the severity of heart failure in patients. NYHA class I is the mildest, indicating that the patient has heart disease but is not limited in physical activity; NYHA class IV is the most severe, indicating that the patient has heart disease, experiences heart failure or angina symptoms at rest, and discomfort increases with any physical activity.

[0121] A total of 252 heart failure patients with a clear NYHA functional class were consecutively randomly enrolled during their outpatient and emergency visits. This classification was determined by the hospital's clinicians based on the patients' comprehensive clinical signs. Among them, there were 18 patients in NYHA class I, 69 patients in NYHA class II, 88 patients in NYHA class III, and 77 patients in NYHA class IV. Blood samples were collected from patients on the day of their visit using lithium heparin vacuum blood collection tubes (BD, CNL20-C0371). After processing, the samples were tested using the NT-IGFBP-4 kit provided in Example 1. Statistical analysis was performed using GraphPad Prism 9.0, and a two-sided significance level of P < 0.05 was considered statistically significant.

[0122] like Figure 7 As shown, the measured concentrations of NT-IGFBP-4 were statistically significant between each pair of the four NYHA groups (I, II, III, and IV). Higher NT-IGFBP-4 concentrations were associated with higher NYHA functional class scores, indicating more severe disease. This demonstrates a significant correlation between NT-IGFBP-4 concentration levels and NYHA functional class, and its measurement can be used to diagnose or assist in the diagnosis of the severity of heart failure.

[0123] Example 6 This embodiment is used to evaluate the predictive value of NT-IGFBP-4 in the prognosis of heart failure.

[0124] This study consecutively included 275 patients hospitalized for acute exacerbations of chronic heart failure. Follow-up lasted 240 days. During this period, 115 patients dropped out, 10 were excluded due to congenital heart disease, malignancy, severe infection, or other abnormal conditions, and 3 were excluded due to non-cardiac readmissions. Ultimately, 147 patients were included in the prognostic study for statistical analysis. The specific procedures for sample inclusion in the prognostic study are detailed below. Figure 8 Fresh blood samples were collected from patients at the beginning of their hospital stay (on the day of admission or the following day) using lithium heparin vacuum blood collection tubes (BD, CNL20-C0371). After processing, the samples were immediately tested using the NT-IGFBP-4 kit provided in Example 1.

[0125] I. Long-term prognosis of heart failure Of the 147 patients, 32 experienced endpoint events within 240 days of follow-up, including 30 cases of centrally-induced readmission and 2 cases of all-cause death. Patients were divided into survivors and non-survivors based on whether an endpoint event occurred.

[0126] NT-IGFBP-4, NT-proBNP, and their combined values ​​were used for prognostic assessment of heart failure within 240 days. ROC curves were used to analyze the predictive value of NT-IGFBP-4 and NT-proBNP alone and in combination for prognostic risk assessment within 240 days in heart failure patients. Specifically, when the two markers were used in combination, a logistic regression was constructed with prognostic outcome as the dependent variable and the values ​​of both markers as independent variables. The predicted probability for each subject was calculated, and ROC analysis was performed using the predicted probabilities to calculate the AUC value. The results are shown in Table 3.

[0127] Table 3

[0128] Based on the above results, the AUC of NT-IGFBP-4 in assessing the long-term (240 days) prognostic risk of heart failure was 0.783, which was superior to NT-proBNP (AUC=0.730) routinely used in clinical heart failure, showing a statistically significant difference. At the highest Youden index, the optimal threshold for NT-IGFBP-4 was 283 ng / mL, and the optimal threshold for NT-proBNP was 3111 ng / L.

[0129] When NT-IGFBP-4 is used alone to assess the prognostic risk of heart failure, the Kaplan-Meier survival curves during the 240-day follow-up period for heart failure patients are as follows: Figure 9As shown. After dividing patients into risk groups based on the optimal threshold of NT-IGFBP-4 at 283 ng / mL, there was a significant difference between the high-risk and low-risk groups (P<0.0001), with a hazard ratio (logrank) of 5.261 (95% Cl, 2.271–12.19). In the high-risk group, 51% of heart failure patients experienced the endpoint event within 240 days of the prognosis.

[0130] For cases where NT-IGFBP-4 and NT-proBNP are used in combination to assess the prognostic risk of heart failure, the Kaplan-Meier survival curves during the 240-day follow-up period for heart failure patients are as follows: Figure 10 As shown. Based on the thresholds for NT-IGFBP-4 and NT-proBNP, heart failure patients were divided into high-, intermediate-, and low-risk groups. The high-risk group had both markers above their thresholds; the intermediate-risk group had one marker above its threshold; and the low-risk group had both markers below their thresholds. Figure 10 As shown, the combined use of NT-IGFBP-4 and NT-proBNP significantly enhanced the prognostic risk stratification ability in heart failure patients. Specifically, in the high-risk group, 59% of heart failure patients experienced the endpoint event within 240 days of the prognosis.

[0131] The above analysis results indicate that NT-IGFBP-4 alone, or in combination with NT-IGFBP-4 and NT-proBNP, can be used to assess or assist in assessing the long-term (within 240 days) prognostic risk of heart failure, demonstrating good predictive value. Furthermore, the combined use of NT-IGFBP-4 and NT-proBNP significantly enhances the prognostic risk stratification ability for heart failure patients.

[0132] II. Short-term prognosis of heart failure Of the 147 patients, 8 experienced endpoint events within 30 days of follow-up, including 6 cases of centrally-induced readmission and 2 cases of all-cause death. Patients were divided into survivors and non-survivors based on whether an endpoint event occurred.

[0133] NT-IGFBP-4, NT-proBNP, and their combined values ​​were used for prognostic assessment of heart failure within 30 days. ROC curves were used to analyze the predictive value of NT-IGFBP-4 and NT-proBNP alone and in combination for prognostic risk within 30 days in heart failure patients. Specifically, when the two markers were used in combination, a logistic regression was constructed with the prognostic outcome as the dependent variable and the values ​​of both markers as independent variables. The predicted probability for each subject was calculated, and ROC analysis was performed using the predicted probabilities to calculate the AUC value. The results are shown in Table 4.

[0134] Table 4

[0135] Based on the above results, the AUC of NT-IGFBP-4 in assessing the short-term (30-day) prognostic risk of heart failure was 0.806, significantly higher than that of NT-proBNP (AUC=0.669) routinely used in clinical heart failure, showing a statistically significant difference. The optimal threshold for NT-IGFBP-4 was 314 ng / mL, and the optimal threshold for NT-proBNP was 3110 ng / L, coinciding with the highest Youden index.

[0136] For cases where NT-IGFBP-4 or NT-proBNP were used alone to assess the prognostic risk of heart failure, the Kaplan-Meier survival curves during the 30-day follow-up period for the heart failure population were as follows: Figure 11 and Figure 12 As shown, after dividing patients into risk groups based on the optimal threshold of 314 ng / mL for NT-IGFBP-4, there was a significant difference between the high-risk and low-risk groups (P<0.0001). In the high-risk group, 21.4% of heart failure patients experienced endpoint events within 30 days, with a hazard ratio (logrank) of 13.50 (95% Cl, 2.239–81.38), while the hazard ratio for NT-proBNP was only 6.929 (95%, 1.733–27.70, P<0.05). During the 30-day short-term follow-up of heart failure patients, NT-IGFBP-4 demonstrated significant prognostic value, and its prognostic effect was significantly better than that of NT-proBNP, which is currently routinely used in heart failure clinical practice.

[0137] For cases where NT-IGFBP-4 and NT-proBNP are used in combination to assess the prognostic risk of heart failure, the Kaplan-Meier survival curves during the 30-day follow-up period for heart failure patients are as follows: Figure 13 As shown. Based on the thresholds for NT-IGFBP-4 and NT-proBNP, heart failure patients were divided into high-, intermediate-, and low-risk groups. The high-risk group had both markers above their thresholds; the intermediate-risk group had one marker above its threshold; and the low-risk group had both markers below their thresholds. Figure 13 As shown, in the combined use of NT-IGFBP-4 and NT-proBNP, 21.7% of heart failure patients in the high-risk group experienced the endpoint event within 30 days of prognosis. The prognostic risk stratification ability of the combination of the two in heart failure patients was similar to that of NT-IGFBP-4 alone, indicating that the efficacy of NT-IGFBP-4 alone is sufficient to support its use in predicting the 30-day short-term prognosis of heart failure patients.

[0138] Example 7 This embodiment is used to evaluate the predictive value of NT-IGFBP-4 in the prognosis of coronary heart disease.

[0139] This study consecutively included 300 patients hospitalized for coronary artery disease. Follow-up lasted 120 days. During this period, 35 patients dropped out and were excluded; 3 patients were excluded because their samples could not support the measurement of all biomarkers; and 15 patients were excluded from the study cohort due to non-cardiac readmissions such as elective follow-up examinations. Ultimately, 247 patients were included in the prognostic study for statistical analysis. The specific procedures for sample inclusion in the prognostic study are detailed below. Figure 14 Fresh blood samples were collected from patients upon admission (on the day of admission or the following day) using lithium heparin vacuum blood collection tubes (BD, CNL20-C0371). After processing, the samples were immediately tested using the NT-IGFBP-4 kit provided in Example 1. Statistical analysis was performed using GraphPad Prism 9.0 and IBM SPSS Statistics (27.0) software. A statistically significant difference was considered to be p < 0.05 for two-sided analysis.

[0140] Of the 247 patients mentioned above, 13 experienced the observed endpoint event within 120 days, while 234 did not. This embodiment uses ROC curve analysis to assess the prognostic risk of NT-IGFBP-4 in patients with coronary artery disease at 120 days, and the results are shown in Table 5.

[0141] Table 5

[0142] The above results indicate that the application of NT-IGFBP-4 to assess the 120 prognostic risk of patients with coronary heart disease has an AUC of 0.708, which is statistically significant (asymptotic significance < 0.05). The optimal threshold at this point is 174 ng / mL (with the highest Youden index).

[0143] See Figure 15 Kaplan-Meier survival curves during a 120-day follow-up period in patients with coronary artery disease showed that, after dividing risk groups based on an optimal threshold of 174 ng / mL for NT-IGFBP-4, there was a significant difference between the high-risk group (above the threshold) and the low-risk group (below the threshold) (P<0.05), with a hazard ratio (logrank) of 5.132 (95% Cl, 1.348–19.54). These results indicate that NT-IGFBP-4 can be used to assess or assist in assessing the prognostic risk of coronary artery disease and has good predictive value.

[0144] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of antibodies that specifically bind to the N-terminal fragment of insulin-like growth factor binding protein-4 in the preparation of reagents for clinical assessment of heart failure, wherein, The N-terminal fragment of insulin-like growth factor binding protein-4 is: the N-terminal fragment obtained by catalytic hydrolysis of insulin-like growth factor binding protein-4 by pregnancy-associated plasma protein A. The heart failure clinical assessment reagent is used to diagnose or assist in the diagnosis of heart failure, or to assess or assist in the assessment of the prognostic risk of heart failure.

2. Use according to claim 1, characterized in that, The N-terminal fragment of the insulin-like growth factor binding protein-4 includes the amino acid sequence shown in SEQ ID NO:

1.

3. Use according to claim 1, characterized in that, The uses also include: using the antibody that specifically binds to the N-terminal fragment of insulin-like growth factor binding protein-4, obtaining the detection result of the N-terminal fragment of insulin-like growth factor binding protein-4 through immunoassay; obtaining the detection result of the N-terminal brain natriuretic peptide precursor, and combining the detection result of the antibody on the N-terminal fragment of insulin-like growth factor binding protein-4, for the diagnosis or auxiliary diagnosis of heart failure, or for the assessment or auxiliary assessment of the prognostic risk of heart failure.

4. The use as described in any one of claims 1 to 3, characterized in that, The diagnosis or auxiliary diagnosis of heart failure is any one of the following (i) or (ii): (i) To diagnose or assist in the diagnosis of whether the subject has heart failure; (ii) To diagnose or assist in the diagnosis of the severity of heart failure in the subject; Preferably, the severity refers to the left ventricular ejection fraction or NYHA functional class.

5. The use as described in any one of claims 1 to 3, characterized in that, The assessment or auxiliary assessment of the prognostic risk of heart failure is any one of the following (iii) or (iv): (iii) Assess or assist in assessing the short-term prognostic risk of patients with heart failure, where short-term refers to no more than 30 days; (iv) Assess or assist in assessing the long-term prognostic risk of patients with heart failure, where long-term refers to more than 30 days and no more than 240 days; Preferably, the prognostic risk refers to the risk of cardiac readmission and / or death.

6. The use as described in any one of claims 1 to 5, characterized in that, The heart failure clinical assessment reagent includes a first reagent for detecting the N-terminal fragment of insulin-like growth factor binding protein-4, the first reagent comprising a capture antibody and a detection antibody, wherein one of the capture antibody and the detection antibody is an antibody that specifically binds to NT-IGFBP-4, and the other is an antibody that specifically binds to NT-IGFBP-4 or full-length IGFBP-4. Preferably, the first reagent is used for a double-antibody sandwich immunoassay (e.g., a double-antibody sandwich chemiluminescent immunoassay). Preferably, the first reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, a quantum dot, or an enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or a microplate.

7. The use as described in claim 6, characterized in that, The heart failure clinical assessment reagent further includes a second reagent for detecting N-terminal pro-brain natriuretic peptide, the second reagent being used for immunoassays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay), preferably a double-antibody sandwich immunoassay (e.g., double-antibody sandwich ELISA or CLIA). Preferably, the second reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, quantum dots, or an enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or a microplate.

8. The use of antibodies that specifically bind to the N-terminal fragment of insulin-like growth factor binding protein-4 in the preparation of clinical assessment reagents for coronary heart disease, wherein, The N-terminal fragment of insulin-like growth factor binding protein-4 is: the N-terminal fragment obtained by catalytic hydrolysis of insulin-like growth factor binding protein-4 by pregnancy-associated plasma protein A. The aforementioned clinical assessment reagent for coronary heart disease is used to assess or assist in assessing the prognostic risk of coronary heart disease.

9. The use as described in claim 8, characterized in that, The N-terminal fragment of the insulin-like growth factor binding protein-4 includes the amino acid sequence shown in SEQ ID NO:

1.

10. The use as described in claim 8, characterized in that, The prognostic risk of coronary artery disease refers to the risk of cardiac readmission and / or death.

11. The use as described in any one of claims 8-10, characterized in that, The coronary heart disease clinical assessment reagent includes a first reagent for detecting the N-terminal fragment of insulin-like growth factor binding protein-4. The first reagent comprises a capture antibody and a detection antibody, wherein one of the capture antibody and the detection antibody is an antibody that specifically binds to NT-IGFBP-4, and the other is an antibody that specifically binds to NT-IGFBP-4 or full-length IGFBP-4. Preferably, the first reagent is used for a double-antibody sandwich immunoassay (e.g., a double-antibody sandwich chemiluminescent immunoassay). Preferably, the first reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, a quantum dot, or an enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or a microplate.

12. A kit comprising a first reagent for detecting an N-terminal fragment of insulin-like growth factor binding protein-4 and a second reagent for detecting an N-terminal precursor of brain natriuretic peptide.

13. The kit of claim 12, wherein the first reagent comprises a capture antibody and a detection antibody, wherein, One of the capture antibody and the detection antibody is an antibody that specifically binds to NT-IGFBP-4, and the other is an antibody that specifically binds to NT-IGFBP-4 or full-length IGFBP-4. Preferably, the first reagent is used for a double-antibody sandwich immunoassay (e.g., a double-antibody sandwich chemiluminescent immunoassay). Preferably, the first reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, a quantum dot, or an enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or a microplate.

14. The kit according to claim 12 or 13, characterized in that, The second reagent is used for immunoassays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay), preferably for double antibody sandwich immunoassays (e.g., double antibody sandwich ELISA or CLIA). Preferably, the second reagent comprises a capture antibody and a detection antibody, wherein the detection antibody is labeled with a detection tag, such as a radiolabel, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, quantum dots, or an enzyme (e.g., alkaline phosphatase); and / or the capture antibody is coated on the surface of a solid carrier, such as magnetic beads or a microplate.

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