Circulating total NT-proBNP (glycosylated and nonglycosylated NT-proBNP) and its ratio to NT-proBNP (nonglycosylated NT-proBNP) in the assessment of atrial fibrillation

Abstract

The present invention provides a method for diagnosing atrial fibrillation in a subject, comprising: a) determining the amount of total NT-proBNP in a sample from a subject; b) determining the amount of non-glycosylated NT-proBNP in a sample from a subject; c) calculating a score of the amounts determined in steps a) and b); d) comparing the calculated score with a reference score; and e) diagnosing atrial fibrillation in the subject.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for diagnosing atrial fibrillation in a subject, comprising the steps of: a) determining the amount of total NT-proBNP in a sample from the subject, b) determining the amount of non-glycosylated NT-proBNP in a sample from the subject, c) calculating a score of the amounts determined in steps a) and b), d) comparing the calculated score with a reference score, and e) diagnosing atrial fibrillation in the subject. [Background technology]

[0002] 2. Background of the Invention Atrial fibrillation (AF) is the most common cardiac arrhythmia. However, AF is often unrecognized by patients, suggesting that medical history is insensitive to the diagnosis of AF in approximately 40% of patients (Kamel H. et al., Curr Atheroscler Rep 2011:13:338-343).

[0003] The standard test for detecting atrial fibrillation is an electrocardiogram (ECG), preferably performed as a 24-hour ECG (Holter ECG). However, a Holter ECG can only detect atrial fibrillation if an arrhythmia occurs during the 24-hour period of the ECG recording.

[0004] Several publications have suggested an association between enhanced levels of biomarkers and atrial fibrillation (Biomarkers in Atrial Fibrillation: Investigating Biologic Plausibility, Cause, and Effect R. Becker Journal of Thrombosis and Thrombolysis 19(1), 71-75, 2005).

[0005] For example, Buettner et al. investigated the association of N-terminal (NT)-proBNP and NT-proANP levels with three atrial fibrillation (AF) progression phenotypes. The natriuretic peptides were shown to show different sensitivity to AF progression phenotypes (Role of NT-proANP and NT-proBNP in patients with atrial fibrillation: Association with atrial fibrillation progression phenotypes, Buttner, Petra et al. Heart Rhythm, Volume 15, Issue 8, 1132-1137).

[0006] WO 2014 / 072500 discloses that NT-proBNP can be used to assess recent atrial fibrillation.

[0007] Chang et al. disclose the use of NTproBNP (and other biomarkers) in the assessment of atrial fibrillation (Afib). BNP and NT-proBNP have been shown to be associated with atrial fibrillation incidence, postoperative atrial fibrillation incidence, and atrial fibrillation prognosis (Chang et al.: Clinical Applications of Biomarkers in Atrial Fibrillation, The American Journal of Medicine, Vol 130, No 12, December 2017).

[0008] Brain natriuretic peptide (BNP) is a 32 amino acid polypeptide. BNP is synthesized as a 134 amino acid pre-prohormone ("pre-proBNP"). The prohormone ("proBNP", 108 aa long) is created by removing the N-terminal signal peptide with a length of 26 amino acids. The prohormone is then cleaved into NT-proBNP (N-terminus of prohormone brain natriuretic peptide, 76 aa long) and biologically active brain natriuretic peptide (BNP). NT-proBNP and BNP are produced in equimolar amounts. Several studies have shown that assays of BNP and NT-proBNP can be reliably used to diagnose heart failure (see, for example, Prontera et al., Clinica Chimica Acta 400 (2009) 70-73).

[0009] B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are peptides produced in the heart in response to wall stretch and increased volume load (see, e.g., Semenow et al., Clinical Chemistry 65:9 (2019) 1070-72).

[0010] Schellenberger et al. reported that the precursor of B-type natriuretic peptide is an O-linked glycoprotein (Schellenberger et al., Arch Biochem Biophys 2006;451). To date, it is known that there are nine known O-glycosylation sites in proBNP and NTproBNP (Halfinger et al.:Unraveling the Molecular Complexity of O-Glycosylated Endogenous (N-Terminal) pro-B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure.Clinical Chemistry 63:1,359-368(2017)).

[0011] Recently, proBNP glycosylation has emerged as a potential regulatory mechanism in the production of amino-terminal (NT)-proBNP and BNP (see, e.g., Vodovar et al., European Heart Journal, Volume 35, Issue 48, 21 December 2014, Pages 3434-3441). Among the nine identified glycan binding sites within the proBNP molecule, glycosylation of the region proximal to the proBNP cleavage site was shown to play a pivotal role in regulating the enzyme-mediated processing of proBNP (Semenow et al., Clinical Chemistry 65:9(2019)1071). Patients with chronic HF had the highest proportion of glycosylated proBNP compared to patients with acute decompensated HF and non-acute decompensated HF (e.g., Vodovar et al.). Semenow et al. hypothesize that in chronic HF, increased release of glycosylated ProBNP, which is not sufficiently converted to BNP, results in relatively small amounts of active hormone despite high measured concentrations of proBNP.

[0012] Rossji et al. investigated the impact of glycosylation on the diagnostic and prognostic accuracy of NT-proBNP in unselected patients with dyspnea and found that removal of the sugar moiety from NTproBNP1-76 by the use of deglycosylating enzymes in plasma samples from patients with dyspnea, especially those with confirmed heart failure, resulted in higher NT-proBNP concentrations. (Helge Rosjo et al.: Influence of Glycosylation on Diagnostic and Prognostic Accuracy of N-Terminal Pro-B-Type Natriuretic Peptide in Acute Dyspnea: Data from the Akershus Cardiac Examination 2. Clinical Chemistry 61:8,1087-1097(2015).

[0013] In a study by Lewis et al., an assay was developed to distinguish between total proBNP (glycosylated + nonglycosylated proBNP), proBNP that is not glycosylated at threonine 71, and proBNP that is not glycosylated in the central region. ProBNP that is not glycosylated at threonine 71 was shown to be decreased with obesity in patients with heart failure (Semenow et al., 2019; Lewis et al., Clin Chem 2019;65:1115-24).

[0014] So far, it has not been evaluated whether the glycosylation of NT-proBNP (or its precursor proBNP) plays a role in atrial fibrillation.Advantageously, in the study underlying the present invention, it has been found that the measurement of both total NT-proBNP and non-glycosylated NT-proBNP, and the calculation of a score based on the amount (such as ratio) of total NT-proBNP and non-glycosylated NT-proBNP allow reliable assessment of atrial fibrillation.Interestingly, the assessment based on two biomarkers, namely total NT-proBNP and non-glycosylated NT-proBNP, is better than the assessment based on the biomarkers when measured alone. Summary of the Invention

[0015] Accordingly, there is provided a method for diagnosing atrial fibrillation in a subject, comprising: a) determining the amount of total NT-proBNP in a sample from a subject; b) determining the amount of non-glycosylated NT-proBNP in a sample from the subject; c) calculating a score of the amounts determined in steps a) and b); d) comparing the calculated score with a reference score. The present invention relates to a method comprising the steps of:

[0016] In some embodiments, the method of the present invention further comprises: e) diagnosing atrial fibrillation in the subject.

[0017] Preferably, step e) is based on the result of the comparison step d). Thus, step e) may be as follows: e) diagnosing atrial fibrillation in the subject based on the results of the comparison in step d).

[0018] The method referred to by the present invention includes a method consisting essentially of the steps mentioned above or a method comprising further steps. Moreover, the method of the present invention is preferably an ex vivo, more preferably an in vitro method. Moreover, said method may comprise several steps in addition to those explicitly mentioned above. For example, further steps may relate to the determination of further markers and / or sample pretreatment or evaluation of the results obtained by the method. The method may be performed manually or may be assisted by automation. Preferably, steps (a), (b), (c), (d) and / or (e) may be assisted in whole or in part by automation, for example by suitable robotic and sensory equipment for the determination in steps (a) and (b), or by computer-implemented calculation in step (c), or by computer-implemented comparison in step (d). The method of the present invention may be computer-implemented. Detailed description of the invention

[0019] As used herein, the term "diagnose" refers to assessing whether the subject referred to in accordance with the method of the present invention suffers from atrial fibrillation (AF).Preferably, as used herein, the expression "diagnose atrial fibrillation" shall be understood as "assisting" or "assisting" the diagnosis of atrial fibrillation.For example, a physician may be assisted in the diagnosis of atrial fibrillation by additional information and / or devices.Actual diagnosis may therefore be performed by a physician.

[0020] As will be understood by those skilled in the art, the diagnosis of the present invention is not intended to be correct for 100% of the subjects tested. The term "diagnose" preferably requires that a correct diagnosis can be made for a statistically significant portion of subjects. Whether said portion is statistically significant can be determined by those skilled in the art without further ado using various well-known statistical evaluation tools, such as, for example, determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. The preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.4, 0.1, 0.05, 0.01, 0.005, or 0.0001.

[0021] The method of the present invention is useful for the diagnosis of atrial fibrillation. The term "atrial fibrillation" (abbreviated as AF or AFib) is well known in the art. As used herein, the term preferably refers to a supraventricular tachyarrhythmia characterized by uncoordinated atrial activity with a consequent deterioration of the mechanical function of the atria. In particular, the term refers to an abnormal heart rhythm characterized by rapid and irregular beating. Atrial fibrillation is associated with the two upper chambers of the heart. In normal heart rhythm, impulses made by the sinoatrial node spread throughout the heart, causing the myocardium to contract and pump blood. In atrial fibrillation, the regular electrical impulses of the sinoatrial node are replaced by irregular and rapid electrical impulses that result in an irregular heartbeat and an increase in atrial volume. The increase in volume leads to the stretching of tissues that release natriuretic peptides. Symptoms of atrial fibrillation are heart palpitations, fainting, shortness of breath, or chest pain. However, even when it occurs, there are often no symptoms. On the electrocardiogram (ECG), atrial fibrillation is characterized by the replacement of consistent P waves by rapid oscillations or fibrillary waves that vary in amplitude, shape, and timing, accompanied by irregular and frequently rapid ventricular responses in the absence of impaired atrioventricular conduction.

[0022] The European Society of Cardiology (ESC) has proposed the following classification system: newly diagnosed AF, paroxysmal AF, persistent AF, long-term persistent and permanent AF (see Hindricks G et al., doi:10.1093 / eurheartj / ehaa 612, which is incorporated by reference in its entirety. See, e.g., Table 4 in the references).

[0023] All people with AF are initially in a category called first-time diagnosed AF. However, subjects may or may not have previously undetected symptoms. If AF persists for more than one year and is accepted by the patient and physician, the subject suffers from permanent AF and no further attempts are made to restore / maintain sinus rhythm. In particular, conversion back to sinus rhythm is not made (or only by medical intervention). If AF lasts for more than seven days, the subject suffers from persistent AF. The subject may require either pharmacological or electrical intervention to terminate the atrial fibrillation. Thus, persistent AF occurs during episodes, but the arrhythmia does not spontaneously revert to sinus rhythm. Paroxysmal atrial fibrillation refers to intermittent episodes of atrial fibrillation that terminate spontaneously (or by intervention) preferably within seven days of onset. In most cases of paroxysmal AF, symptoms last less than 24 hours. Episodes of paroxysmal atrial fibrillation often terminate spontaneously, i.e., without medical intervention. Paroxysmal AF is often asymptomatic and underdiagnosed (asymptomatic AF). The preferred length of episode of the present invention is less than 48 hours, 24 hours or 12 hours (paroxysmal AF). Thus, as used herein, the term "paroxysmal atrial fibrillation" is defined as an episode of AF that is self-terminating, preferably in less than 48 hours, more preferably in less than 24 hours, and most preferably in less than 12 hours. Preferably, said episode is recurrent. Furthermore, it is envisaged that the episode is self-terminating in less than 6 hours.

[0024] Both persistent and paroxysmal AF may recur within weeks or months, whereby the distinction between paroxysmal and persistent AF is made by ECG recording. AF is considered recurrent if the patient has had two or more episodes. AF, especially recurrent AF, is called paroxysmal if the arrhythmia terminates spontaneously. AF is called persistent if it persists for more than 7 days.

[0025] In some embodiments of the method of the present invention, the atrial fibrillation diagnosed is paroxysmal or persistent atrial fibrillation. In some embodiments of the method, the atrial fibrillation is persistent atrial fibrillation.

[0026] According to the present invention, it is assumed that the atrial fibrillation to be diagnosed is ongoing atrial fibrillation, and thus a subject suffering from atrial fibrillation exhibits symptoms of atrial fibrillation at the time of the test (or more precisely at the time the test sample is obtained).

[0027] The term "sample" refers to a sample of a body fluid, a sample of separated cells, or a sample from a tissue or organ. Body fluid samples can be obtained by well-known techniques and preferably include blood, plasma, serum or urine samples, more preferably blood, plasma or serum samples. Tissue or organ samples can be obtained from any tissue or organ, for example by biopsy. Separated cells may be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. For example, cell, tissue or organ samples may be taken from cells, tissues or organs that express or produce a biomarker.

[0028] In some embodiments of the methods of the present invention, the sample is a blood sample (ie, a whole blood sample), a serum sample or a plasma sample. In some embodiments of the methods of the present invention, the sample is a right atrial appendage tissue sample.

[0029] As used herein, a "subject" is preferably a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits and rodents (e.g., mice and rats). In some embodiments, the subject is a human. The subject may be male or female. The terms "patient" and "subject" are used interchangeably herein.

[0030] In one embodiment, the subject is a female subject. In one embodiment, the subject is a male subject.

[0031] In some embodiments, the subject may have at least one risk factor for atrial fibrillation, e.g., hypertension, such as hypertension requiring antihypertensive medication, heart failure, such as heart failure AHA stages A-C, or a history of stroke.

[0032] In some embodiments, the subject being tested is 50 years of age or older, such as 60 years of age or older. In some embodiments, the subject is 70 years of age or older.

[0033] Preferably, the subject to be tested is a subject suspected of suffering from atrial fibrillation. A subject suspected of suffering from atrial fibrillation is a subject who shows at least one symptom of atrial fibrillation and / or has shown at least one symptom of atrial fibrillation before carrying out the method for evaluating atrial fibrillation. The symptoms are usually transient, occurring within a few seconds and may disappear just as quickly. Symptoms of atrial fibrillation include dizziness, fainting, shortness of breath, and especially heart palpitations. Thus, the at least one symptom of atrial fibrillation is selected from dizziness, fainting, shortness of breath, and especially heart palpitations. Preferably, the subject has shown at least one symptom of atrial fibrillation within 6 months, more preferably within 1 month, even more preferably within 2 weeks, and most preferably within 1 week prior to obtaining the sample. In particular, it is assumed that the subject has shown at least one symptom of AF within 2 days prior to obtaining the sample. It is also assumed that the subject has shown at least one symptom of AF within 24 hours, or even within 12 hours prior to obtaining the sample. Thus, the subject is suspected of having atrial fibrillation, ie, symptoms of atrial fibrillation, within one of these time periods.

[0034] According to the present invention, it is assumed that the subject suspected to suffer from atrial fibrillation has a medical history of atrial fibrillation, i.e., a known medical history of atrial fibrillation.Therefore, the subject shall have been previously diagnosed as suffering from atrial fibrillation, i.e., before carrying out the method of the present invention (particularly before obtaining a sample from the subject).Furthermore, the subject may have previously had undiagnosed symptoms of atrial fibrillation.

[0035] According to step a) of the method of the present invention, the amount of total NT-proBNP is determined, i.e. measured, in a sample from a subject. According to step b), the amount of non-glycosylated NT-proBNP is determined in a sample from a subject (e.g. a blood, serum or plasma sample). It should be understood that steps a) and b) may be performed in any order. Also, each step may be performed simultaneously.

[0036] The term "NT-proBNP" (N-terminal fragment of pro-brain natriuretic peptide) is well known in the art. As used herein, this term refers to a 76 amino acid N-terminal fragment of pro-brain natriuretic peptide (proBNP), a secreted protein that functions as a cardiac hormone after cleavage. Methods for measuring NT-proBNP are commercially available and are used in clinical practice, such as those sold under the name Roche Elecsys® proBNP II.

[0037] The sequence of human NT-proBNP is well known in the art and has already been described in detail in the prior art, for example in WO 02 / 089657, WO 02 / 083913, Bonow 1996, New Insights into the cardiac natriuretic peptides. Circulation 93:1946-1950. Preferably, human NT-proBNP has the amino acid sequence shown in SEQ ID NO:1.

[0038] As mentioned above, NT-proBNP and its precursor proBNP can be O-glycosylated. Overviews of the O-glycosylation of NT-proBNP and proBNP are provided, for example, by Schellenberger et al. and Halflinger et al., both of which are incorporated herein by reference in their entirety (Schellenberger et al., Arch Biochem Biophys 2006;451; Halfinger et al., Clinical Chemistry 63:1,359-368(2017)).

[0039] The term "O-linked glycosylation" is well known in the art (also referred to herein as "glycosylation"). O-linked glycosylation is the attachment of a sugar molecule, i.e., a carbohydrate, to a serine or threonine residue. O-linked glycosylation is a post-translational modification that occurs after a protein is synthesized. How NT-proBNP and proBNP are glycosylated is described, for example, in Schellenberger et al., and Halflinger et al. (cited above).

[0040] An overview regarding the O-glycosylation sites of NT-proBNP is provided herein below. The following sequence is the human NT-proBNP sequence (herein SEQ ID NO: 1 serves as the reference sequence). underline is drawn. His Pro Leu Gly Ser Pro Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly 1 5 10 15 Leu Gln Glu Gln Arg Asn His Leu Gln Gly Lys Leu Ser Glu Leu Gln 20 25 30 Val Glu Gln Thr Ser Leu Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr 35 40 45 Gly Val Trp Lys Ser ArgGluValAla Thr Glu Gly Ile Arg Gly His 50 55 60 Arg Lys Met Val Leu Tyr Thr Leu Arg Ala Pro Arg (SEQ ID NO: 1) 65 70 75

[0041] Thus, human NT-proBNP (set forth in SEQ ID NO:1) contains at least the following glycosylation sites: T36, S37, S44, T48, S53, T58 and T71.

[0042] Preferably, as used herein, the term "unglycosylated NT-proBNP" refers to NT-proBNP that is not glycosylated (i.e., not O-glycosylated) at one or more positions selected from the group consisting of T36, S37, S44, T48, S53, T58 and T71 of human NT-proBNP. Thus, at least one of the following amino acid residues of human NT-proBNP: T36, S37, S44, T48, S53, T58 and T71 is not glycosylated, i.e., does not contain O-glycosylation.

[0043] In some embodiments, the term "unglycosylated NT-proBNP" refers to NTproBNP in which at least two of the aforementioned amino acid residues (i.e., T36, S37, S44, T48, S53, T58 and T71) are not glycosylated. In some embodiments, the term "unglycosylated NT-proBNP" refers to NTproBNP in which at least three of the aforementioned amino acid residues are not glycosylated. In some embodiments, the term "unglycosylated NT-proBNP" refers to NTproBNP in which at least three, at least four, at least five, at least six, or all of the aforementioned amino acid residues are not glycosylated. In some embodiments, the term "unglycosylated NT-proBNP" refers to NTproBNP in which at least the serine residue at position 44 (i.e., S44) is not glycosylated. Thus, unglycosylated NT-proBNP may not be glycosylated at position S44 (i.e., Ser44).

[0044] In a preferred embodiment, determining the amount of nonglycosylated NT-proBNP in a sample from a subject comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects nonglycosylated NT-proBNP. Preferably, the antibody or antigen-binding fragment thereof that specifically detects nonglycosylated NT-proBNP specifically binds to an epitope of NT-proBNP, the epitope including the glycosylation site, but not glycosylated at the glycosylation site. The complex formed between the antibody (or fragment) and the biomarker should be proportional to the amount of nonglycosylated NT-proBNP.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof that specifically detects non-glycosylated NT-proBNP specifically binds to an epitope of NT-proBNP, said epitope comprising the T36 amino acid residue, said T36 amino acid residue being unglycosylated. Preferably, said antibody or fragment does not substantially bind to NT-proBNP comprising a glycosylated T36 amino acid residue.

[0046] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, wherein the epitope comprises the S37 amino acid residue, and wherein said S37 amino acid residue is unglycosylated. Preferably, the antibody or fragment does not substantially bind to NT-proBNP that comprises a glycosylated S37 amino acid residue.

[0047] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, the epitope comprising the S44 amino acid residue, and the S44 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not substantially bind to NT-proBNP comprising a glycosylated S44 amino acid residue. In some embodiments, the epitope of the antibody or antigen-binding fragment thereof comprises amino acid residues 42-46 of human NT-proBNP (as set forth in SEQ ID NO:1, see also FIG. 2).

[0048] In a preferred embodiment, the antibody that specifically detects non-glycosylated NT-proBNP is the monoclonal antibody MAB1.21.3 disclosed in WO2004099253, or an antibody comprising the six CDRs of said antibody. Furthermore, it is envisaged to use an antigen-binding fragment of said antibody.

[0049] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, said epitope comprising the T48 amino acid residue, and said T48 amino acid residue is unglycosylated. Preferably, the antibody or fragment does not substantially bind to NT-proBNP comprising a glycosylated T48 amino acid residue.

[0050] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, which epitope comprises the S53 amino acid residue, and said S53 amino acid residue is unglycosylated. Preferably, the antibody or fragment does not substantially bind to NT-proBNP that comprises a glycosylated S53 amino acid residue.

[0051] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, said epitope comprising the T58 amino acid residue, and said T58 amino acid residue is unglycosylated. Preferably, the antibody or fragment does not substantially bind to NT-proBNP comprising a glycosylated T58 amino acid residue.

[0052] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, said epitope comprising the T71 amino acid residue, and said T71 amino acid residue is unglycosylated. Preferably, the antibody or fragment does not substantially bind to NT-proBNP comprising a glycosylated T71 amino acid residue.

[0053] Preferably, as used herein, the term "glycosylated NT-proBNP" refers to NT-proBNP that is glycosylated (i.e., O-glycosylated), preferably at one or more positions (i.e., amino acid residues) selected from the group consisting of T36, S37, S44, T48, S53, T58 and T71 of human NT-proBNP. Thus, at least one of the following amino acid residues of human NT-proBNP is glycosylated, i.e., comprises an O-glycosylation: T36, S37, S44, T48, S53, T58 and T71.

[0054] Further suitable epitopes are disclosed in FIG.

[0055] In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least two of the aforementioned amino acid residues (i.e., T36, S37, S44, T48, S53, T58 and T71) are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least three of the aforementioned amino acid residues are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least three, at least four, at least five, at least six, or all of the aforementioned amino acid residues are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least the serine residue at position 44 (i.e., S44) is glycosylated.

[0056] "Total amount of NT-proBNP" is preferably the amount of glycosylated and non-glycosylated NT-proBNP. Thus, the term refers to the sum of the amount of glycosylated NT-proBNP and non-glycosylated NT-proBNP.

[0057] Preferably, determining the amount of total NT-proBNP comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP. More preferably, said antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP binds to a region of human NT-proBNP that cannot be glycosylated. Thus, said antibody (or fragment thereof) should specifically bind to a region of NT-proBNP, in particular a region of human NT-proBNP, that does not have a glycosylation site, i.e. an O-glycosylation site. The complex formed between the antibody (or fragment) and the biomarker should be proportional to the amount of total NT-proBNP.

[0058] In particular, said antibody (or fragment thereof) shall specifically bind to a region of NT-proBNP that does not have the glycosylation sites T36, S37, S44, T48, S53, T58 or T71 (of human NT-proBNP). For example, the first 35 amino acid residues, i.e. the N-terminal amino acid residues 1-35, are known to have no O-glycosylation sites. Preferably, an antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP binds to an epitope present within the first 35 amino acids of NT-proBNP, more preferably, to an epitope present within the first 20 amino acids of NT-proBNP, most preferably, to an epitope present within amino acid residues 10-20 of human NT-proBNP. In a preferred embodiment, said antibody epitope or antigen-binding fragment thereof comprises amino acid residues 13-16 of human NT-proBNP. The sequence of human NT-proBNP is shown above (see SEQ ID NO: 1).

[0059] In a preferred embodiment, the antibody that specifically detects total NT-proBNP is the monoclonal antibody MAB17.3.1 disclosed in WO2004099253, or an antibody comprising the six CDRs of said antibody. Furthermore, it is envisaged to use an antigen-binding fragment of said antibody.

[0060] The term "antibody" is known in the art. As used herein, the term refers to any immunoglobulin (Ig) molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains. As used herein, the term "antibody" also includes antigen-binding fragments of antibodies. As used herein, an antigen-binding fragment of an antibody is one that is capable of specifically binding to an antigen (especially NT-proBNP as described above). Thus, an antigen-binding fragment of an antibody is a fragment that retains the ability of a (full-length) antibody to specifically bind to an antigen (e.g., non-glycosylated NT-proBNP or total NT-proBNP). An antibody fragment preferably comprises a portion of a full-length antibody, preferably a variable domain thereof, or at least an antigen-binding site thereof. In one embodiment, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a Facb fragment, a F(ab')2 fragment, a scFv fragment, and a Fv fragment. For example, the antigen-binding fragment is a F(ab')2 fragment. Methods for producing antigen-binding fragments are well known in the art. For example, fragments can be produced by enzymatic cleavage of the antibody of the present invention.Furthermore, fragments can be produced synthetically or by recombinant techniques.Fab fragments are preferably produced by papain digestion, pepsin digestion and partial reduction of antibodies to produce Fab' fragments, pepsin digestion to produce F(ab')2 fragments, and plasmin digestion to produce facb fragments.Fv or scFv fragments are preferably produced by molecular biology techniques.

[0061] The antibody according to the method of the present invention may be a polyclonal or monoclonal antibody. In a preferred embodiment, the antibody is a monoclonal antibody. The term "monoclonal antibody" is well known in the art. As used herein, the term preferably refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody, which variants are generally present in minor amounts. The monoclonal antibodies of the present invention may be produced by the well-known hybridoma method described in Kohler and Milstein, Nature, 256:495 (1975). They may also be produced by recombinant DNA methods. In some embodiments, the monoclonal antibody is selected from the group consisting of sheep monoclonal antibodies, mouse monoclonal antibodies, rabbit monoclonal antibodies, goat monoclonal antibodies, horse monoclonal antibodies, and chicken monoclonal antibodies. In some embodiments, the monoclonal antibody is a mouse monoclonal antibody.

[0062] The antibodies (or fragments) used in steps a) and b) of the method of the invention may be used in a sandwich assay as capture antibodies in combination with at least one other antibody that binds to a different, i.e. additional, NT-proBNP epitope, preferably said at least one other antibody that binds to a region of NT-proBNP that is not glycosylated as described above.

[0063] Antibodies may be used in sandwich assays. "Sandwich assays" are one of the most useful and commonly used assays, and encompass several variations of sandwich assay techniques. For example, in a typical assay, an unlabeled (capture) binding agent is immobilized or can be immobilized on a solid substrate, and the sample to be tested is contacted with the capture binding agent. After a suitable incubation period, sufficient time to allow the formation of a binding agent-biomarker complex, a second (detection) binding agent labeled with a reporter molecule capable of generating a detectable signal is added and incubated for a sufficient time to allow the formation of another complex of binding agent-biomarker-labeled binding agent. Any unreacted material may be washed away, and the presence of the biomarker is determined by observing the signal generated by the reporter molecule bound to the detection binding agent. The results may be qualitative, by simple observation of a visible signal, or may be quantified, for example, by comparison with a control sample containing a known amount of the biomarker to be determined (as a standard or calibrator as described elsewhere herein).

[0064] The incubation step of typical sandwich assay can be modified as necessary and appropriate.Such modifications include, for example, simultaneous incubation, in which two or more binding agents and biomarkers are co-incubated.For example, both the sample to be analyzed and the labeled binding agent are added to the immobilized capture binding agent at the same time.It is also possible to first incubate the sample to be analyzed and the labeled binding agent, and then add the antibody that is bound to the solid phase or can be bound to the solid phase.

[0065] The complex formed between the specific binding agent and the biomarker shall be proportional to the amount of the biomarker present in the sample. It will be understood that the specificity and / or sensitivity of the applied binding agent defines the extent to which the proportion of at least one marker contained in the sample can be specifically bound. Details of how the measurement can be carried out are also described elsewhere in this specification. The amount of the complex formed shall be converted into the amount of the biomarker, which reflects the amount actually present in the sample.

[0066] As used herein, the term "amount" encompasses the absolute amount of total NT-proBNP or non-glycosylated NT-proBNP, the relative amount or concentration of said total NT-proBNP or non-glycosylated NT-proBNP, as well as any value or parameter that correlates thereto or can be derived therefrom. Such values ​​or parameters include intensity signal values ​​derived from any specific physical or chemical property obtained from the peptide by direct measurement, e.g. intensity values ​​in mass or NMR spectra. Furthermore, all values ​​or parameters obtained by indirect measurement as specified elsewhere herein are encompassed, e.g. response levels determined from a biological readout system in response to the peptide or intensity signals obtained from specifically bound ligands. It is to be understood that values ​​that correlate with the above-mentioned amounts or parameters can also be obtained by all standard mathematical operations. According to a preferred embodiment of the present invention, the determination of the "amount" is performed by the disclosed system, whereby a computing device determines the "amount" based on contacting and measuring steps performed by one or more analyzer units of said system.

[0067] In step c) of the method of the invention, a score of the amounts determined in steps a) and b), ie the amount of total NT-proBNP and the amount of non-glycosylated NT-proBNP, is calculated.

[0068] As used herein, the term "calculating" refers to evaluating a score, which is based on the amount of total NT-proBNP and the amount of non-glycosylated NT-proBNP determined in a sample of a subject. For example, it is envisaged to calculate a score, i.e. a single score, based on the amount of total NT-proBNP and the amount of non-glycosylated NT-proBNP, and compare this score with a reference score. The calculated score combines information on the amount of total NT-proBNP and the amount of non-glycosylated NT-proBNP. Furthermore, biomarkers may be weighted in the score according to their contribution to establishing a diagnosis. This score may be considered as a classifier parameter for diagnosing atrial fibrillation. In particular, this score shall allow the diagnosis of AF based on comparison with a reference score. The reference score is preferably a value, in particular a cut-off value, that allows to distinguish between subjects suffering from AF and subjects not suffering from AF.

[0069] Preferably, the score is a ratio, i.e. the ratio of the amount of total NT-proBNP and the amount of non-glycosylated NT-proBNP. The ratio calculated in step c) is therefore compared to a reference ratio. In one embodiment, the ratio is the ratio of the amount of total NT-proBNP to the amount of non-glycosylated NT-proBNP. In another embodiment, the ratio is the ratio of the amount of non-glycosylated NT-proBNP to the amount of total NT-proBNP.

[0070] In step d) of the method of the invention, the score calculated in step c) is to be compared with a reference score, for example the calculated ratio is to be compared with a reference ratio.

[0071] As used herein, the term "comparing" includes comparing the calculated scores for samples from test subjects with appropriate reference sources as specified elsewhere herein. The comparison is preferably assisted by automation. For example, a suitable computer program may be used that includes an algorithm for comparing the calculated subject scores with the reference scores. Such computer programs and algorithms are well known in the art. Notwithstanding the above, the comparison may also be performed manually. The computer program may further evaluate the results of the comparison, i.e., automatically provide the desired evaluation, i.e., diagnostic result, in a suitable output format. Said diagnostic result may preferably serve as an aid for establishing the final diagnosis of atrial fibrillation, for example, by a physician.

[0072] The calculating and / or comparing steps may be performed using a computer comprising a processing unit.

[0073] Based on the comparison of the calculated score with the reference score, it shall be possible to evaluate whether the subject suffers from atrial fibrillation.For example, the result of the comparison may be given as raw data, or in some cases as an indicator in the form of a word, phrase, symbol or number that may indicate a certain diagnosis.It is necessary to select the reference score so that the difference or identity between the calculated score and the calculated score allows to identify whether the subject belongs to the group of subjects suffering from atrial fibrillation.This method allows either the exclusion (exclusion) or identification (exclusion) of subjects suffering from atrial fibrillation.The difference of the score, i.e. increase or decrease, as used herein, is preferably a statistically significant difference.

[0074] Preferably, the reference score, such as the reference ratio, allows the subject to distinguish whether or not the subject suffers from atrial fibrillation.Preferably, the diagnosis is made by evaluating whether the subject's score is above or below the reference score.It is not necessary to provide an exact reference score.Relevant reference scores can be obtained by correlating the sensitivity and specificity and sensitivity / specificity for any score.The reference score that brings about high sensitivity brings about low specificity, and vice versa.

[0075] In some embodiments, the reference score is derived from a sample from a subject (or a sample group of subjects) known to suffer from atrial fibrillation.

[0076] In some embodiments, the reference score is derived from a sample obtained from a subject (or a sample group of subjects) known not to be afflicted by atrial fibrillation.

[0077] A preferred diagnostic algorithm is provided below.

[0078] As described above, the score calculated in step c) may be a ratio. In one embodiment, the ratio is the ratio of the amount of total NT-proBNP to the amount of non-glycosylated NT-proBNP. In this case, a ratio (i.e., the calculated ratio) lower than the reference ratio indicates a subject suffering from atrial fibrillation. A ratio higher than the reference ratio indicates a subject not suffering from atrial fibrillation.

[0079] In another embodiment, the calculated ratio is the ratio of the amount of non-glycosylated NT-proBNP to the amount of total NT-proBNP. In this case, a ratio (i.e., the calculated ratio) greater than the reference value indicates a subject suffering from atrial fibrillation. A ratio lower than the reference ratio indicates a subject not suffering from atrial fibrillation.

[0080] In a preferred embodiment of the method of the present invention, the method further comprises the step of recommending an appropriate therapy if atrial fibrillation is diagnosed. Alternatively, the method further comprises the step of initiating an appropriate therapy if atrial fibrillation is diagnosed.

[0081] As used herein, the term "recommend" refers to establishing a treatment proposal applicable to a subject. However, it should be understood that the term does not include any actual application of treatment. The recommended treatment depends on the result of the diagnosis provided by the method of the present invention. The above recommendation step can also be preferably automated. Preferably, the diagnosis obtained by the method of the present invention, i.e., the diagnostic result of the method, is used to search a database containing recommendations of treatment measures for each possible diagnosis result.

[0082] In one embodiment, the recommended or initiated treatment is the administration of at least one anticoagulant, i.e. anticoagulant therapy. Anticoagulant therapy is preferably a therapy aimed at reducing or preventing blood clotting and associated strokes. In a preferred embodiment, the at least one anticoagulant is selected from the group consisting of heparin, coumarin derivatives (i.e. vitamin K antagonists), in particular warfarin or dicoumarol, oral anticoagulants, in particular dabigatran, rivaroxaban or apixaban, tissue factor pathway inhibitor (TFPI), antithrombin III, factor IXa inhibitors, factor Xa inhibitors, inhibitors of factors Va and VIIIa, and thrombin inhibitors (anti-IIa type). In some embodiments, the at least one anticoagulant is selected from the group consisting of direct factor Xa inhibitors, direct thrombin inhibitors, and PAR-1 antagonists. It is therefore envisaged that the subject (if diagnosed as suffering from atrial fibrillation) will take at least one of the aforementioned drugs.

[0083] In some embodiments, the anticoagulant is a direct factor Xa inhibitor, such as apixaban, rivaroxaban, darexaban, or edoxaban. In some embodiments, the anticoagulant is a direct thrombin inhibitor, such as dabigatran. In some embodiments, the anticoagulant is a PAR-1 antagonist, such as vorapaxar or atopaxar.

[0084] In another embodiment, the recommended or initiated therapy is cardioversion. Thus, the patient may undergo cardioversion. Cardioversion is a medical procedure that uses electricity or drugs to convert cardiac arrhythmias to a normal rhythm.

[0085] In some embodiments, the cardioversion is cardioversion, such as synchronized cardioversion.

[0086] In some embodiments, the cardioversion is drug-induced cardioversion.Therefore, at least one antiarrhythmic drug is administered.In some embodiments, at least one antiarrhythmic drug is selected from amiodarone, flecainide, ibutilide, lidocaine, procainamide, propafenone, quinidine and tocainide.

[0087] Advantageously, it has been further shown that the ratio of non-glycosylated NT-proBNP / total-NT-proBNP is low in reports of heart failure (see examples).The score referred to herein therefore makes it possible to distinguish between atrial fibrillation and heart failure as causes of NT-proBNP elevation, and therefore makes it possible to distinguish between atrial fibrillation and heart failure.

[0088] The definitions and explanations given herein above preferably apply mutatis mutandis to the following methods of the invention.

[0089] The method of the present invention may also be implemented as a computer-implemented invention. In one embodiment, one or more steps, such as the comparing step and / or the calculating step, are performed by a computer including a processing unit (i.e., a computer). In another embodiment, all steps are performed by a computer equipped with a processing unit.

[0090] Accordingly, the present invention provides a computer-implemented method for diagnosing atrial fibrillation in a subject, comprising: (a) in a processing unit, (a1) a value for the amount of total NT-proBNP in a sample from the subject; and (a2) the amount of non-glycosylated NT-proBNP in a sample derived from the subject; Receiving (b) processing the values ​​received in step (a) by the processing unit, (b1) calculating a score of the values ​​(a1) and (a2) received in step a); (b2) processing the values, including comparing the calculated scores to reference scores; and (c) optionally providing said diagnosis via an output device, said diagnosis being based on the results of step b); The present invention relates to a computer-implemented method,

[0091] In some embodiments, the processing unit is comprised in a computer.

[0092] In some embodiments, step b) further comprises retrieving, in the processing unit, a reference score from a memory, i.e. a reference score suitable for the diagnosis of AF.

[0093] In one embodiment of the method of the present invention, the information about diagnosis (due to the last step of the method of the present invention) is provided via a display configured to present the evaluation.Thus, as described elsewhere herein, information can be provided whether the subject suffers from atrial fibrillation or not.Furthermore, a recommendation of appropriate treatment can be displayed.As described elsewhere herein, various treatment measures can be recommended.In this case, treatment options can be displayed on the display.

[0094] In one embodiment of the method of the invention, the method may comprise the further step of transferring information relating to the evaluation of the method of the invention to the subject's electronic medical record.

[0095] Alternatively, the assessment performed in the last step of the method of the present invention may be printed by a printer, the printout containing information regarding whether the patient is at risk or not at risk and / or a recommendation for appropriate therapeutic action.

[0096] The present invention further relates to the use of i) total NT-proBNP and non-glycosylated NT-proBNP or ii) at least one agent that specifically binds to non-glycosylated NT-proBNP and at least one agent that specifically binds to total NT-proBNP, as biomarkers, for diagnosing atrial fibrillation. Preferably, said use is an in vitro use, i.e. carried out in a sample from a subject.

[0097] Preferred agents are disclosed elsewhere herein (eg, antibodies or antigen-binding fragments thereof that bind to specific epitopes within NT-proBNP).

[0098] Finally, the present invention relates to a kit comprising at least one agent that specifically binds to non-glycosylated NT-proBNP and at least one agent that specifically binds to total NT-proBNP.

[0099] As used herein, the term "kit" refers to a collection of the aforementioned means provided, for example, separately or in a single container. The container may include instructions for carrying out the method of the present invention.

[0100] List of embodiments In the following, preferred embodiments are summarized. The definitions and explanations given herein above preferably apply mutatis mutandis to the following embodiments.

[0101] 1. A method for diagnosing atrial fibrillation in a subject, comprising: a) determining the amount of total NT-proBNP in a sample from said subject; b) determining the amount of non-glycosylated NT-proBNP in a sample from said subject; c) calculating a score of the amounts determined in steps a) and b); d) comparing the calculated score to a reference score; and e) diagnosing atrial fibrillation in said subject. A method comprising:

[0102] 2. The method of embodiment 1, wherein the sample is a blood, serum, or plasma sample.

[0103] 3. The method of embodiments 1 and 2, wherein the subject is a human subject.

[0104] 4. The method of any one of embodiments 1-3, wherein the subject is suspected of suffering from atrial fibrillation.

[0105] 5. The method of embodiment 4, wherein the subject suspected of suffering from atrial fibrillation has a medical history of atrial fibrillation.

[0106] 6. The method of any one of embodiments 1-5, wherein the non-glycosylated NT-proBNP is not glycosylated at one or more positions selected from the group consisting of T36, S37, S44, T48, S53, T58 and / or T71 of human NT-proBNP.

[0107] 7. The method of embodiment 6, wherein the non-glycosylated NT-proBNP is not glycosylated at least at position S44.

[0108] 8. The method of any one of embodiments 1 to 7, wherein determining the amount of nonglycosylated NT-proBNP comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects nonglycosylated NT-proBNP.

[0109] 9. The method of embodiment 8, wherein the epitope of said antibody or antigen-binding fragment thereof comprises amino acid residues 42-46 of human NT-proBNP.

[0110] 10. The method of any one of embodiments 1-9, wherein the amount of total NT-proBNP is the amount of glycosylated and non-glycosylated NT-proBNP.

[0111] 11. The method according to any one of the preceding embodiments, wherein determining the amount of total NT-proBNP comprises contacting said sample with an antibody or an antigen-binding fragment thereof that specifically detects NT-proBNP.

[0112] 12. The method of embodiment 11, wherein said antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP binds to a region of human NT-proBNP that does not have an O-glycosylation site.

[0113] 13. The method of embodiment 12, wherein the antibody or antigen-binding fragment thereof binds to an epitope present in the first 35 amino acids of NT-proBNP.

[0114] 14. The method of embodiment 13, wherein the epitope of the antibody or antigen-binding fragment thereof comprises amino acid residues 13-16 of human NT-proBNP.

[0115] 15. The method of any one of the preceding embodiments, wherein the score is a ratio.

[0116] 16. The method of embodiment 15, wherein the ratio is the ratio of the amount of total NT-proBNP to the amount of nonglycosylated NT-proBNP.

[0117] 17. The method of embodiment 16, wherein a ratio lower than the reference ratio is indicative of the subject suffering from atrial fibrillation.

[0118] 18. The method of embodiment 15, wherein the ratio is the ratio of the amount of nonglycosylated NT-proBNP to the amount of total NT-proBNP.

[0119] 19. The method of embodiment 18, wherein a ratio higher than the reference ratio is indicative of the subject suffering from atrial fibrillation.

[0120] 20. The method according to any one of the preceding embodiments, wherein the atrial fibrillation is paroxysmal or persistent atrial fibrillation.

[0121] 21. A computer-implemented method for diagnosing atrial fibrillation in a subject, comprising: (a) in a processing unit, (a1) a value for the amount of total NT-proBNP in a sample from the subject; and (a2) the amount of non-glycosylated NT-proBNP in a sample derived from the subject; Receiving (b) processing the values ​​received in step (a) by the processing unit, (b1) calculating a score of the values ​​(a1) and (a2) received in step a); (b2) processing the values ​​received in step (a), including comparing the calculated scores with reference scores; and (c) optionally providing said diagnosis via an output device, said diagnosis being based on the results of step b); 4. A computer-implemented method comprising:

[0122] 22. The use of i) total NT-proBNP and non-glycosylated NT-proBNP, or ii) at least one agent that specifically binds to non-glycosylated NT-proBNP and at least one agent that specifically binds to total NT-proBNP, as biomarkers, for diagnosing atrial fibrillation.

[0123] 23. A kit comprising at least one agent that specifically binds to non-glycosylated NT-proBNP and at least one agent that specifically binds to total NT-proBNP. [Brief description of the drawings]

[0124] [Figure 1A] Differential expression of NPPB in right atrial appendage tissue: A) persistent Afib; B) paroxysmal Afib. [Figure 1B] Differential expression of NPPB in right atrial appendage tissue: A) persistent Afib; B) paroxysmal Afib. [Diagram 2] Schematic of O-glycosylation sites in the proBNP epitopes. The sequence of human NT-proBNP is underlined twice. In the studies underlying the present invention, two antibodies were used: one antibody that binds to epitope aa13-16 of NT-proBNP (for total NT-proBNP) and one antibody that binds to epitope aa42-46 of NT-proBNP for NT-proBNP, i.e., non-glycosylated NT-proBNP. Other antibodies that bind to other epitopes may be used as well, for example, antibodies from Hytest that bind to the epitopes as shown in the figure. Antibodies are also disclosed in US Patent Application Publication No. 20090163415. [Figure 3A] Differences between patients with ongoing atrial fibrillation and those in sinus rhythm based on total NT-proBNP. [Figure 3B] Differences between patients with ongoing atrial fibrillation and those in sinus rhythm based on total NT-proBNP. [Figure 4A] Differences between patients with ongoing atrial fibrillation and those in sinus rhythm based on nonglycosylated NT-proBNP. [Figure 4B] Differences between patients with ongoing atrial fibrillation and those in sinus rhythm based on nonglycosylated NT-proBNP. [Figure 5A] Difference between patients with ongoing atrial fibrillation and those in sinus rhythm based on the ratio [nonglycosylated NT-proBNP] / [total NT-proBNP]. [Figure 5B] Difference between patients with ongoing atrial fibrillation and those in sinus rhythm based on the ratio [nonglycosylated NT-proBNP] / [total NT-proBNP]. EXAMPLES

[0125] The present invention is illustrated merely by the following examples, which should not be construed in any way as limiting the scope of the invention.

[0126] Example 1: Differential expression of human NPPB in right atrial appendage tissue (mapping study) Right atrial appendage tissue was sampled during open-chest surgery for CABG or valve surgery. Evidence of AF or SR (control, sinus rhythm) was obtained intraoperatively using simultaneous endocardial-epicardial high-density activation mapping. Tissue samples were taken during surgery. AF and control patients were matched for sex, age, and comorbidities.

[0127] Atrial tissue samples were prepared for: ● Paroxysmal AF patients; n = 14 patients Patients with persistent AF; n = 8 patients ● SR control patients; n = 27 patients

[0128] The differential expression of NPPB was determined by RNAseq analysis applying the algorithms RSEM and DESEQ2. The results are shown in Figure 1 [A) Persistent Afib B) Paroxysmal Afib].

[0129] As shown in Figure 1, in the analyzed right atrial appendage tissues of patients with paroxysmal and persistent AF, expression of NPPB was found to be upregulated in sinus rhythm compared to control patients. The highest levels were observed in patients with persistent AF. These data support the hypothesis of the atrial appendage dependence of NT-proBNP and its increase with disease severity.

[0130] Example 2: Detection of NT-proBNP The amounts of total NT-proBNP and NT-proBNP were measured by sandwich assay. Total NT-proBNP was measured using an antibody against amino acids 13-16 of NT-proBNP (monoclonal antibody 17.3.1) as capture antibody. NT-proBNP was measured using an antibody against amino acids 42-46 of NT-proBNP (monoclonal antibody 1.21.03) as capture antibody using the Roche Elecsys® proBNP II NT-proBNP assay according to the manufacturer's instructions. This antibody detects NT-proBNP but not the O-glycosylation position S44.

[0131] For both assays, a detection antibody that binds to amino acids 27-31 (monoclonal antibody 18.4.34, see Figure 2) was used.

[0132] The epitopes are shown in FIG.

[0133] Higher values ​​were obtained with the non-glycosylated total NT-proBNP assay due to the elimination of the effects caused by the inhibition of detection of O-glycosylation at position S44. In clinical practice, the present invention increases the sensitivity, improving specificity and increasing sensitivity for the detection of various entities.

[0134] A ratio of total NT-proBNP to non-glycosylated NT-proBNP can be formed, which allows for normalization.

[0135] Example 3: Detection of Atrial Fibrillation in the Biomarker Substudy of the GISSI-AF Trial In the biomarker substudy of the GISSI-AF trial, blood samples were collected at study enrollment and at 6 and 12 months of follow-up. For details of the GISSI-AF trial, see main publication: GISSI-AF Investigators, New Engl J Med 2009;360:1606-17. For further details of the biomarker substudy, see Latini R et al., J Intern Med 2011;269:160-71.

[0136] Total and NT-proBNP values ​​from plasma samples were obtained at baseline for 382 patients. After 24 weeks, 38 of 360 patients had paroxysmal atrial fibrillation. After 52 weeks, 48 ​​of 357 patients had atrial fibrillation.

[0137] NT-proBNP and total NT-proBNP were measured in a selected atrial fibrillation subcohort from the GISSI AF study. Elevated circulating NT-proBNP and total NT-proBNP levels were observed in samples from subjects with ongoing atrial fibrillation versus controls. [Table 1]

[0138] The sample size considered consisted of 382 patients with baseline NTproBNP measurements. The variable SR (sinus rhythm) vs. AF (atrial fibrillation) was recorded from the CRF variable "ritmo all'ECG".

[0139] Conclusions: Measurement of total NT-proBNP and NT-proBNP showed that, upon analysis, the combination of both parameters yielded a much better diagnostic value than each single marker. [Table 2]

[0140] ROC analysis was performed to assess the strength of association between the tested biomarkers and ongoing AF (i.e., biomarkers assayed while the AF rhythm was recorded). At 6 and 12 months, the AUC observed for the ratio of NT-proBNP / total NT-proBNP was higher compared to the AUC for each biomarker alone: ​​ratio of NT-proBNP / total NT-proBNP (AUC6M=0.93 and AUC12M=0.91), total NT-proBNP (AUC6M=0.78 and AUC12M=0.77), NT-proBNP (AUC6M=0.87 and AUC12M=0.86).

[0141] In Table 2 and Figures 3, 4 and 5, the ratio of NT-proBNP / total NT-proBNP proved to be a better predictor of ongoing AF prevalence / progression than any one of the natriuretic peptides alone.

[0142] The GISSI AF study further observed that the ratio of NT-proBNP / total NT-proBNP was associated with incident / recurrent AF (p=0.058).

[0143] In conclusion, we found that NT-proBNP was less glycosylated in patients with ongoing AF compared with patients in sinus rhythm. Determination of the NTproBNP / total NTproBNP ratio is suitable to further improve the diagnostic accuracy for each natriuretic peptide alone. [Table 3]

[0144] To assess the strength of association between test biomarkers (i.e., biomarkers assayed while AF rhythms were recorded) and ongoing AF in female and male patients, receiver operating characteristic (ROC) analyses were performed separately for male and female study participants. For the ratio of NT-proBNP / total NT-proBNP, the AUCs observed at 6 and 12 months were higher in female patients compared to male patients: NTproBNP / total NTproBNP ratio in female patients (AUC6m=0.94 and AUC12m=0.95), NTproBNP / total NTproBNP ratio in male patients (AUC6M=0.92 and AUC12M=0.92).

[0145] As shown in Table 3, the ratio of NTproBNP / total NT-proBNP was found to perform particularly well in detecting ongoing AF in the subgroup of female participants in the study. The ratio of NT-proBNP / total-NTpro BNP was approximately 0.15 in Afib (Table 2), but was higher in reports of heart failure (0.19-0.31; Vodovar N et al. European Heart Journal (2014) 35, 3434-3441), but the present invention using the ratio of NT-proBNP / total-NTpro BNP in AFib allows for a better distinction between AFib vs. HF as the source of NT-proBNP elevation, and therefore, AFib vs. heart failure.

[0146] In conclusion, for the detection of AF in women, the ratio NTproBNP / total NTpro-BNP may be very useful.

[0147] In the GISSIAF study, patients were included who had: clinically diagnosed HF or LVEF <40% (11%), and the following moderate to severe comorbidities: history of stroke (4%), diabetes mellitus (13%), history of hypertension (84%), and documented CAD (11%). The incidence of AF was not significantly different between patients with any of the observed comorbidities and those without each comorbidity.

[0148] In conclusion, patients with incident or history of AF were found to have a lower degree of NTproBNP glycosylation compared with patients in sinus rhythm.

[0149] Overall, it was observed that the NTproBNP / total NTproBNP ratio is capable of improving the general diagnosis of AF in patients with a history of AF or other risk factors for incident AF recurrence.

Claims

1. 1. A method for determining atrial fibrillation in a subject, comprising: a) determining the amount of total NT-proBNP in a sample from said subject, b) determining the amount of non-glycosylated NT-proBNP in a sample from said subject; c) calculating a score from the amounts determined in step a) and step b); and d) comparing the calculated score with a reference score; A method comprising:

2. The method of claim 1 , wherein the sample is a blood, serum, or plasma sample and / or the subject is a human subject.

3. The method of claim 1 or 2, wherein the subject is suspected of suffering from atrial fibrillation.

4. 4. The method of claim 3, wherein the subject suspected of having atrial fibrillation has a medical history of atrial fibrillation.

5. The method according to any one of claims 1 to 4, wherein the non-glycosylated NT-proBNP is not glycosylated at one or more positions selected from the group consisting of T36, S37, S44, T48, S53, T58 and / or T71 of human NT-proBNP.

6. The method according to claim 5, wherein the non-glycosylated NT-proBNP is not glycosylated at least at position S44.

7. The method according to any one of claims 1 to 6, wherein determining the amount of nonglycosylated NT-proBNP comprises contacting the sample with an antibody or antigen-binding fragment thereof which specifically detects nonglycosylated NT-proBNP.

8. The method of claim 7, wherein the epitope of said antibody or antigen-binding fragment thereof comprises amino acid residues 42 to 46 of human NT-proBNP.

9. The method according to any one of claims 1 to 8, wherein the amount of total NT-proBNP is the amount of glycosylated and non-glycosylated NT-proBNP.

10. The method of claim 9, wherein determining the amount of total NT-proBNP comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP.

11. The method of claim 10, wherein the antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP binds to a region of human NT-proBNP that does not have an O-glycosylation site.

12. the antibody or antigen-binding fragment thereof binds to an epitope present in the first 35 amino acids of NT-proBNP; The method of claim 11, wherein the epitope of the antibody or antigen-binding fragment thereof comprises amino acid residues 13 to 16 of human NT-proBNP.

13. The method of any one of claims 1 to 12, wherein the score is a ratio.

14. a) said ratio is the ratio of the amount of said total NT-proBNP to the amount of said non-glycosylated NT-proBNP, or The method according to claim 13, wherein said ratio is the ratio of the amount of said non-glycosylated NT-proBNP to the amount of said total NT-proBNP.

15. The method according to any one of claims 1 to 13, wherein the atrial fibrillation is paroxysmal or persistent atrial fibrillation.

16. 1. A computer-implemented method for diagnosing atrial fibrillation in a subject, comprising: (a) in a processing unit, (a1) a value for the amount of total NT-proBNP in a sample from said subject, and (a2) a value for the amount of non-glycosylated NT-proBNP in a sample derived from said subject; Receiving (b) processing the values ​​received in step (a) by the processing unit, comprising: (b1) calculating a score of the values ​​(a1) and (a2) received in step a); (b2) comparing the calculated score with a reference score; processing the values ​​received in step (a), 4. A computer-implemented method comprising:

17. (c) providing a diagnosis via an output device, said diagnosis being based on the results of step b); 20. The computer-implemented method of claim 16, comprising:

18. i) the use of total NT-proBNP and non-glycosylated NT-proBNP, or ii) the use of at least one agent that specifically binds to non-glycosylated NT-proBNP and at least one agent that specifically binds to total NT-proBNP, as biomarkers for diagnosing atrial fibrillation.

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