Assay for long form of cardiac troponin t

Abstract

The present invention relates to an in vitro method for determining the likelihood of acute myocardial infarction in a subject based on certain long forms of cardiac troponin T. The invention also relates to a kit for use in the method.

Description

Technical Field

[0001] The present invention relates to an assay for determining the likelihood of acute myocardial infarction (MI) in a subject and a kit for use in the assay. Background Art

[0002] High-sensitivity cardiac troponin assays are key elements in the diagnostic workup of acute coronary syndromes. The widespread use of troponin measurements has led to a situation where mild troponin elevations are frequently found in daily practice and are not usually due to type 1 myocardial infarction (MI). Other conditions such as chronic kidney disease, atrial fibrillation, strenuous exercise, and heart failure may cause transient or persistent elevations in plasma troponin levels, which can present diagnostic challenges and lead to repeated use of diagnostic coronary angiography or “overdiagnosis” of MI.

[0003] During MI, cardiac troponin is released from ischemic myocardium as a combination of full-size troponin and troponin fragments, which are subsequently proteolytically degraded in the serum in a time-dependent manner. The determinants and mechanisms of cardiac troponin release in other clinical situations are not fully understood. Free, fragmented troponin in the cytoplasm can cross cell membranes that are leaky but not irreversibly damaged. Commercially available high-sensitivity troponin T (cTnT) tests also detect these small fragments, thus leading to an erroneous diagnosis of MI. Therefore, the need for accurate diagnostic troponin testing has been identified, especially for patient populations in which elevated troponin levels are common. Summary of the invention

[0004] In one aspect, the invention relates to an in vitro assay for determining the likelihood of acute myocardial infarction (MI) in a subject according to claim 1 .

[0005] In another aspect, the present invention relates to a kit according to claim 17 for determining the likelihood of acute MI in a subject.

[0006] In some further aspects, the present invention relates to the use of the kit listed in claim 24 for determining the likelihood of acute MI in a subject, and the use of the kit listed in claim 25 for distinguishing patients with MI from patients who show elevated standard cTnT levels for reasons other than MI.

[0007] Further aspects, embodiments and details are set out in the following figures, detailed description, examples and dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings are included to provide a further understanding of the present invention and constitute a part of this specification, which illustrate embodiments of the present invention and together with the description help explain the principles of the present invention. In the drawings:

[0009] Figure 1 Schematic representation of cTnT molecular forms detectable by conventional standard cTnT assay and novel long cTnT assay according to one embodiment of the present invention. The black tracer antibody is marked with an asterisk, while the capture antibody is white.

[0010] Figure 2 The long / standard cTnT ratios in patients with ESRD, NSTEMI, and STEMI are shown. Long cTnT was measured using an immunoassay using three tracer antibodies.

[0011] Figure 3 Standard cTnT, long cTnT, and long / standard cTnT ratio in patients with ESRD and NSTEMI patients with a delay between symptom onset and sample collection of ≤24 hours are shown. Long cTnT was measured using an immunoassay using three tracer antibodies.

[0012] Figure 4 The relationship between the long / standard cTnT ratio and the time from symptom onset to blood sampling in patients with NSTEMI and STEMI is shown. The decline in the ratio follows a typical exponential decay function with an elimination half-life of 13.9 hours (95% confidence interval is shown as a dashed line, elimination constant 0.0499, standard error 0.0075, r=0.613). Long cTnT was measured using an immunoassay using three tracer antibodies.

[0013] Figure 5 Long (left) and standard (right) cTnT before and immediately after hemodialysis in the ESRD group are shown. Long cTnT was measured by immunoassay using three tracer antibodies.

[0014] Figure 6 Receiver operating characteristic curves are shown, depicting the discriminatory ability of the cTnT ratio (long / standard) (solid line) and standard cTnT (dashed line) between ESRD and NSTEMI (all patients: cTnT ratio AUC 0.906 (CI95% 0.837-0.975) and standard cTnT AUC 0.688 (CI95% 0.575-0.800) [Panel A]; NSTEMI patients with a delay of ≤24 hours between symptom onset and sample collection: cTnT ratio AUC 0.955 (CI95% 0.899-1.0) and standard cTnT AUC 0.609 (CI95% 0.454-0.764) [Panel B]). Long cTnT was measured using an immunoassay using three tracer antibodies.

[0015] Figure 7 Shown are standard cTnT, long cTnT, and long / standard cTnT ratio in patients with ESRD and NSTEMI patients with a delay between symptom onset and sample collection ≤ 24 hours when long cTnT was measured by immunoassay using only one tracer antibody. DETAILED DESCRIPTION

[0016] Before describing the present invention, it should be understood that the present disclosure is not strictly limited to any specific compositions, reagents, antibodies, devices, protocols or methods described herein, as these may vary. It should also be understood that the terminology used herein is only used to describe specific embodiments and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0017] It should also be noted that unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] It should also be noted that, for clarity, certain features of the disclosure described in the context of a separate embodiment may also be provided in combination in a single embodiment. On the contrary, for brevity, the various features of the disclosure described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. In addition, even if not repeated, any feature, detail or embodiment disclosed in the context of the method provided herein is also applicable to the kit provided herein, and vice versa. Accordingly, even if not repeated, any such feature, detail or embodiment is also applicable to the various uses of the kit.

[0019] As used herein and in the appended claims, the singular forms "a," "an," and "the" mean one or more. Therefore, unless otherwise indicated, singular nouns also have the meaning of the corresponding plural nouns, and vice versa. Therefore, the terms "a," "an," "one or more," and "at least one" can be used interchangeably.

[0020] The term "and / or" in phrases such as "X and / or Y" should be understood as "X and Y" or "X or Y" and should be considered to provide clear support for both meanings or either meaning.

[0021] The terms "comprising," "including," and "having" are used interchangeably and are intended to be interpreted in a non-exclusive manner, ie, to allow for the presence of items, components or elements not expressly described.

[0022] As used herein, the term "myocardial infarction" (MI) refers to a very dangerous condition, commonly known as a heart attack, which is caused by insufficient blood flow to the heart muscle. Acute MI is caused by acute blockage of the coronary arteries. Common symptoms of MI include, for example, chest pain or discomfort, abnormal heartbeat, nausea, vomiting, shortness of breath, and fatigue. MI is diagnosed by electrocardiogram (ECG) and the presence or absence of serological markers (especially cardiac troponin).

[0023] As used herein, the term "ST-segment elevation myocardial infarction" (STEMI) refers to a type of MI that has a pattern on the ECG called ST-segment elevation. Measurement of cardiac troponin is not necessary for diagnosis, although it is usually analyzed when the situation permits.

[0024] As used herein, the term "non-ST-segment elevation myocardial infarction" (NSTEMI) refers to a type of MI that is diagnosed based on symptoms consistent with MI and elevated cardiac troponins but without ECG changes indicative of STEMI.

[0025] As used herein, the term "end-stage renal disease" (ESRD), also referred to as end-stage renal failure, is the final, long-term stage of chronic kidney disease (CKD). Symptoms of CKD and ESRD include, for example, chest pain, nausea, vomiting, shortness of breath, fatigue and weakness. Elevated levels of high-sensitivity cardiac troponin T are common in CKD and ESRD patients. Therefore, it is challenging to diagnose NSTEMI in CKD and ESRD patients.

[0026] As used herein, the term "cardiac troponin T" (cTnT) refers to one of the subunits of human cardiac troponin, which is a myofibrillar protein present in cardiac tissue and plays an important role in regulating muscle contraction by regulating the binding of tropomyosin and actin. Cardiac troponin T is considered a strong marker for acute myocardial infarction. However, elevated cTnT levels are also present in patients with impaired renal function and can also be seen in other conditions.

[0027] Human cardiac troponin T is encoded by the TNNT2 gene. At least ten different isoforms of cTnT have been identified, which are produced by the alternative splicing of mRNA transcripts. The main isoform found in normal adult heart tissue is isoform 6, also known as TNT3 (UniProtKB / Swiss-Prot entry P45379-6). The calculated molecular weight of isoform 6 is about 34.6kDa, and its amino acid sequence is shown in SEQ ID NO:1, and it is composed of 288 amino acids.

[0028] It should be understood that although isoform 6 is used herein as a reference cTnT, the present invention is not limited thereto. Thus, for example, any and all references to certain cTnT fragments refer not only to fragments of isoform 6, but also encompass corresponding fragments of other cTnT isoforms present in the human heart after birth. Likewise, the amino acid sequence set forth in SEQ ID NO: 1 represents a reference cTnT sequence from isoform 6. Thus, any reference to a specific subsequence or amino acid residue in SEQ ID NO: 1 encompasses corresponding subsequences or amino acid residues in other related cTnT isoforms.

[0029] Cardiac TnT is cleaved in vivo to produce cTnT fragments of varying sizes. The major cTnT cleavage sites have been documented to be located between amino acid residues corresponding to amino acids 68 and 69 in SEQ ID NO:1, within the C-terminal cleavage region around amino acid residues corresponding to amino acids 189-223 in SEQ ID NO:1 with multiple cleavage sites, and between amino acid residues 287 and 288 in SEQ ID NO:1. N-terminal cleavage produces an N-terminal short fragment (~8 kDa, aa 1-68 in SEQ ID NO:1) and a C-terminal long fragment (~29 kDa, aa 69-287 or aa 69-288 in SEQ ID NO:1). C-terminal cleavage produces an N-terminal long fragment (~22-30 kDa) and a C-terminal short fragment (~8-15 kDa). The exact amino acid boundaries of the N-terminal long fragment and the C-terminal short fragment vary due to the presence of multiple potential cleavage sites in the C-terminal cleavage region. Cleavage at two major cleavage sites generates N-terminally and C-terminally truncated mid-fragments (~14-20 kDa, the major 16 kDa fragment represents aa69-189 in SEQ ID NO: 1). The molecular weights indicated refer to the estimated molecular weight of cTnT isoform 6 based on Western blot analysis. The amino acid boundaries indicated also refer to the amino acid boundaries of cTnT isoform 6. Therefore, it should be understood that the molecular weight and amino acid boundaries may vary depending on the cTnT isoform in question.

[0030] As used herein, the term "long cTnT" refers to forms of cTnT that are not cleaved at the major C-terminal cleavage region corresponding to amino acids 189-223 of SEQ ID NO: 1 and include a C-terminal region corresponding to amino acids 205-215 of SEQ ID NO: 1. These forms may be referred to herein as the first group of cTnT molecules. Thus, the term includes both the complete or full-length cTnT (~37 kDa) and the C-terminal long fragment (~29 kDa), as well as possible other fragments that include the above regions. However, the first group of cTnT molecules does not include any C-terminally truncated fragments, i.e., fragments that are cleaved at the C-terminal region corresponding to amino acids 189-223 of SEQ ID NO: 1, including a C-terminal short fragment (~8-15 kDa), an N-terminal short fragment (~8 kDa), and a medium fragment (~14-20 kDa) that is truncated at both the N-terminus and the C-terminus.

[0031] As used herein, the term "intact cTnT" refers to a substantially full-length cTnT molecule (~37 kDa) that is not cleaved or is cleaved only at the C-terminal cleavage site located between the last two amino acids corresponding to amino acids 287 and 288 in SEQ ID NO: 1. In other words, "intact cTnT" has an amino acid sequence corresponding to amino acids 1-287 or amino acids 1-288 in SEQ ID NO: 1.

[0032] As used herein, the term "standard cTnT" refers to a form of cTnT that can be detected by the gold standard high sensitivity cTnT (hs-cTnT) assay commercially available from Roche Diagnostics or any corresponding cTnT assay. The Roche assay works according to the sandwich principle, in which the detection antibody (M7) and the capture antibody (M11.7) are allowed to bind to cTnT, forming a sandwich complex. Since the two antibodies bind tightly to each other in the central part of the cTnT molecule, the assay is able to detect all cTnT isoforms and fragments thereof that contain the epitope sequences corresponding to these antibodies. Therefore, the term "standard cTnT" includes complete cTnT (~37 kDa), C-terminal long fragment (~29 kDa), N-terminal long fragment (~22-30 kDa), and the middle fragment (~14-20 kDa) that is truncated at both the N-terminus and the C-terminus. In other words, the term includes all other cTnT fragments except the N-terminal short fragment (~8 kDa) and the C-terminal short fragment (~8-15 kDa). For simplicity, the cTnT forms denoted as "standard cTnT" may also be collectively referred to as the second group of cTnT molecules.

[0033] As used herein, the term "long / standard cTnT ratio" refers to the calculated ratio of the measured concentration of long cTnT to the measured concentration of standard cTnT, i.e., the long cTnT concentration divided by the standard cTnT, and optionally multiplied by 100% to express the ratio as a percentage value. In other words, the "long / standard cTnT ratio" defines the proportion of standard cTnT in a given sample that is composed of long cTnT. For example, a ratio of 0.5 and 50% both mean that half of the standard cTnT is composed of long cTnT. However, affected by the calibration of the assay and possible assay-specific interferences, in some cases, the ratio may be higher than 1 or 100%.

[0034] As used herein, the term "standard / long cTnT ratio" refers to the calculated ratio of the measured concentration of standard cTnT to the measured concentration of long cTnT, i.e., the standard cTnT concentration divided by the long cTnT, and optionally multiplied by 100% to express the ratio as a percentage value. In other words, the "standard / long cTnT ratio" defines the relative proportion of standard cTnT to long cTnT in a given sample. For example, a ratio of 2 and 200% both mean that the concentration of standard cTnT is twice that of long cTnT, and half of the standard cTnT is composed of long cTnT.

[0035] As used herein, the term "third group of cTnT molecules" refers collectively to "C-terminal cTnT molecules", i.e., all cTnT forms comprising the C-terminal portion of cTnT (i.e., the region ranging from the C-terminal cleavage region (aa 189-223 of SEQ ID NO: 1) to the C-terminal end (aa 287 or 288 of SEQ ID NO: 1)). Such molecules include complete cTnT (~37 kDa), C-terminal long fragment (~29 kDa), and C-terminal short fragment (~8-15 kDa), but do not include N-terminal long fragment (~22-30 kDa), N-terminal short fragment (~8 kDa), or medium fragment (~14-20 kDa) with both N-terminal and C-terminal truncations.

[0036] As used herein, the term "diagnosis" refers generally to (but not limited to) a process intended to determine or assess the probability of whether a subject suffers from a disease or condition (e.g., acute myocardial infarction). This also means including situations where it has not yet been finally determined that a disease or condition exists and further diagnostic testing is required. In such embodiments, the method itself cannot determine the presence or absence of the disease or condition, but may indicate that further diagnostic testing is required or that further diagnostic testing is beneficial. Diagnostic test results can also exclude the presence of a given disease or condition without a final diagnosis. In some cases, diagnostic test results can also be used to strengthen the diagnosis that has been performed using other indicators. Therefore, the present method can be combined with one or more other diagnostic methods to finally determine the presence or absence of the disease or condition in the subject. Such other diagnostic methods are well known to those skilled in the art, including but not limited to ECG.

[0037] As used herein, the term "subject" refers to an animal subject, preferably a mammalian subject, and more preferably a human subject. Herein, the terms "subject," "individual," and "patient" are interchangeable, especially when referring to human subjects.

[0038] As used herein, the term "sample" refers in particular to a blood sample obtained from a subject, such as a serum or plasma sample. However, a plasma sample, such as a heparin plasma sample, an EDTA plasma sample, etc., is preferred, since it is known that thrombin present in normal serum can cleave cTnT at the N-terminal cleavage site between the amino acid residues corresponding to amino acids 68 and 69 in SEQ ID NO: 1. Generally, obtaining a sample from a subject is not part of the assays or methods disclosed herein, and therefore, as in vitro assays or methods, they need to be performed using a sample previously collected from a patient.

[0039] As used herein, "sensitivity" is a measure of the ability of a marker (eg, cTnT) to detect a particular disease or condition (eg, MI). In other words, sensitivity represents the probability of a positive test result occurring in a subject with the disease or condition.

[0040] As used herein, a "true positive" (TP) refers to a test result that correctly classifies a subject with a particular disease or condition as a subject with the disease or condition. Likewise, a "true negative" (TN) refers to a test result that correctly classifies a non-diseased subject as a non-diseased subject.

[0041] As used herein, "specificity" is a measure of the ability of a marker to detect the absence of a particular disease or condition. In other words, specificity represents the probability of a negative test result occurring in a subject who does not suffer from the disease or condition.

[0042] As used herein, a "false positive" (FP) refers to a test result in which a non-diseased subject is incorrectly classified as a diseased subject (i.e., a subject with the disease or condition). Similarly, a "false negative" (FN) refers to a test result in which a subject with a particular disease or condition is incorrectly classified as a non-diseased subject.

[0043] As used herein, the term "accuracy" (also referred to as "diagnostic validity") refers to the proportion of correctly classified subjects (TP+TN) among all subjects (TP+TN+FP+FN).

[0044] Receiver Operating Characteristic (ROC) curve can be used to show the balance between the sensitivity and specificity of markers, as known to those skilled in the art. The horizontal X-axis of ROC curve represents 1-specificity, which increases with the increase of false positive rate. The vertical Y-axis of the curve represents sensitivity, which increases with the increase of true positive rate. Therefore, for selected specific cutoff value (i.e. threshold value), the value of specificity and sensitivity can be determined. In other words, the data points on the ROC curve represent the ratio of true positive and false positive classification at various decision boundaries. When the true positive rate is close to 1.0 and the false positive rate is close to 0.0, the best results can be obtained. However, when the cutoff value is changed to improve specificity, sensitivity is usually reduced, and vice versa.

[0045] The area under the ROC curve, commonly referred to as AUC, is a measure of the utility of a marker in correctly identifying disease subjects (i.e., subjects suffering from a disease or condition). Therefore, the AUC value can be used to determine the effectiveness of a test. An area of ​​1.0 represents a perfect test; an area of ​​0.5 represents a worthless test. A traditional rough guide to classifying the accuracy of a diagnostic test is as follows: AUC values ​​of 0.9 to 1.0 represent tests with excellent diagnostic ability, AUC values ​​of 0.80 to 0.90 represent tests with good diagnostic ability, AUC values ​​of 0.70 to 0.80 represent tests with fair diagnostic ability, AUC values ​​of 0.60 to 0.70 represent tests with poor diagnostic ability, and AUC values ​​of 0.50 to 0.60 represent tests with failed diagnostic ability.

[0046] As used herein, the term "binding molecule" refers broadly to any agent that specifically binds to its target epitope. The term includes, but is not limited to, antibodies as well as non-antibody scaffold formats such as, but not limited to, affibodies and aptamers.

[0047] As used herein, the term "antibody" generally refers to an immunoglobulin structure comprising two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, including but not limited to polyclonal, monoclonal and recombinant antibodies of isotypes IgA, IgD, IgE, IgG and IgM and subtypes thereof. Means and methods for producing antibodies are readily available in the art.

[0048] Antibodies may exist as intact immunoglobulins or as any of a number of well-characterized antigen-binding fragments or single-chain variants thereof, all of which are encompassed herein by the term "antibody". Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments (i.e., single-chain variable fragments) and nanobodies (i.e., monomeric variable domains of camelid heavy chain antibodies), as well as such fragments when engineered to form fusions with the Fc region. Antigen-binding fragments and single-chain variants may be produced by recombinant DNA techniques, or by enzymatic or chemical separation of immunoglobulins as is well known in the art.

[0049] As used herein, the term "epitope" refers generally to the portion of a target protein that is specifically recognized by an antibody or other binding molecule. As is well known in the art, some epitopes may be discontinuous, i.e., composed of several small fragments that are scattered in the amino acid sequence, and may even be in different subunits or polypeptide chains of the protein, but are close in the three-dimensional structure of the protein. Sometimes, when the amino acids of different proteins become very close, an epitope can be formed from them, for example due to the formation of a complex. Such an epitope cannot be defined by a linear amino acid sequence.

[0050] As used herein, the terms "specifically bind", "binds specifically" or "has specificity" refer to the discriminatory binding of a binding molecule to its target sequence or epitope such that the binding molecule does not substantially cross-react with non-target sequences, i.e., does not have significant non-specific binding.

[0051] As will be readily appreciated by those skilled in the art, binding molecules need not be 100% complementary to their target sequences, as long as they still specifically bind to the target epitope. Thus, even if some amino acids at the N-terminal or C-terminal end of the epitope region are missing (e.g. due to cleavage of the cTnT molecule), the binding molecule can still bind to its target epitope.

[0052] As used herein, the term "quantification" and its corresponding expressions refer to quantifying or measuring the amount of cTnT or a group of cTnT molecules (such as long cTnT or standard cTnT) in a sample. The term "amount" is interchangeable with the terms "level" and "concentration" and can refer to absolute quantification or relative quantification. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention relates broadly to a new generation of troponin assays, and more particularly to assays for detecting long cTnT in a sample, and in particular for determining the ratio of long cTnT to standard cTnT in a sample. The present assays have improved specificity for acute MI compared to conventional high-sensitivity standard cardiac troponin T (hs-cTnT) assays, since standard cardiac troponin T is commonly elevated in conditions other than MI, including, for example, acute or chronic renal dysfunction, atrial fibrillation, and strenuous exercise.

[0055] The assay comprises quantifying the amount of long cTnT in the sample by contacting the sample with at least one first binding molecule capable of specifically binding to the C-terminal portion of cTnT, more specifically to the region corresponding to amino acids 201-288, preferably amino acids 223-288 of SEQ ID NO: 1, and at least one second binding molecule capable of specifically binding to the portion towards the N-terminus, more specifically to the region corresponding to amino acids 1-200, preferably amino acids 1-190, 69-200, 69-190, 164-200 or 164-190 of SEQ ID NO: 1. Surprisingly, the first binding molecule may also be selected from binding molecules capable of specifically binding to cardiac troponin I (cTnI; SEQ ID NO: 2), cardiac troponin C (cTnC; SEQ ID NO: 3) or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed with each other. The binding of both the at least one first binding molecule and the at least one second binding molecule to the same troponin T molecule distinguishes the long cTnT molecule from other common cTnT fragments that may be present in the sample, including the most prominent N-terminal long fragment, the N-terminal and C-terminal truncated middle fragment, and the C-terminal short fragment. The concentration of long cTnT in the sample is related to the level of binding reaction involving both the first binding molecule and the second binding molecule. In some embodiments, the number of the first binding molecule and the second binding molecule varies independently of each other, usually 1 to 3, that is, usually 1, 2 or 3.

[0056] In some other embodiments, the assay comprises quantifying the amount of long cTnT in a sample by contacting the sample with at least one first binding molecule and at least one second binding molecule, wherein the first binding molecule is capable of specifically binding to the complete C-terminal cleavage region of cTnT, more specifically, to the region corresponding to amino acids 195-220 of SEQ ID NO: 1, and its binding to its epitope is severely impaired due to cleavage of the cleavage region; the second binding molecule is capable of specifically binding to: the portion toward the N-terminus, more specifically, to the region corresponding to amino acids 1-194, preferably 1-190, 69-194, 69-190, 164-194 or 164-190 of SEQ ID NO: 1; the portion toward the C-terminus, more specifically, to the region corresponding to amino acids 221-288, preferably 221-287, 224-287 or 224-288 of SEQ ID NO: 1; cTnI (SEQ ID NO: 2); cTnC (SEQ ID NO: 3); NO:3); or when long cTnT is complexed with cTnI and cTnC or when cTnI is complexed with cTnC. By definition, since the C-terminal cleavage region of long cTnT is intact, such a first binding molecule can correctly distinguish long cTnT from other forms of cTnT molecules (i.e., cTnT fragments cleaved at the C-terminal cleavage region) that may be present in the sample whose long cTnT content is to be quantified. Therefore, in these embodiments, the binding of both at least one first binding molecule and at least one second binding molecule to the same troponin T molecule (or cTnI or cTnC complexed with the troponin T molecule) distinguishes the long cTnT molecule from other common cTnT fragments that may be present in the sample, including the most prominent N-terminal long fragment, the N-terminal and C-terminal truncated middle fragments, and the C-terminal short fragment. The concentration of long cTnT in the sample is related to the level of binding reaction involving both the first binding molecule and the second binding molecule. In some embodiments, the number of the first binding molecule and the second binding molecule varies independently of each other, and is usually 1 to 3, ie, usually 1, 2 or 3.

[0057] In some embodiments, the first binding molecule is a capture molecule, such as a capture antibody, and the second binding molecule is a tracer molecule, such as a tracer antibody, or vice versa. In some embodiments, more than one (usually 1, 2, or 3) capture and / or tracer molecules can be used to improve detection. In some embodiments, a capture molecule is used in combination with one, two, or three tracer molecules.

[0058] In some embodiments, the first binding molecule is specific for a cTnT epitope corresponding to amino acids 223-242 of SEQ ID NO: 1. In some further embodiments, the second binding molecule is specific for a cTnT epitope selected from the group consisting of an epitope corresponding to amino acids 67-86, amino acids 119-138, amino acids 125-130, amino acids 132-151, amino acids 136-147, amino acids 145-164, amino acids 171-190 of SEQ ID NO: 1. In some other embodiments, the first binding molecule can be an anti-cTnC antibody targeting amino acids 1-100 or the full-length protein, or an anti-cTnI antibody specific for a cTnI epitope selected from the group consisting of: amino acids 2-16, 14-23, 19-23, 19-29, 19-36, 23-32, 23-41, 24-30, 25-41, 41-49, 42-50, 87-91, 26-41, 27-36, 35-38, 42-50, 66-75, 84-94, 87-91, 88-91, 118-127, 131-146, 137-148, 170-179, 187-193, 191-197, 196-210 corresponding to SEQ ID NO:2. Both anti-cTnC and cTnT antibodies are commercially available from a variety of suppliers, including, for example, HyTest, Novus Biologicals, and Thermo Fisher Scientific.

[0059] Furthermore, antibodies specific for the epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed with each other are also commercially available, for example from HyTest.

[0060] In the assay according to some embodiments, the first binding molecule is a capture molecule, preferably a capture antibody, which is specific for a cTnT epitope corresponding to amino acids 223-242 of SEQ ID NO: 1, and the second binding molecule is a tracer molecule, preferably a tracer antibody, which is specific for a cTnT epitope selected from the epitopes corresponding to amino acids 67-86, 119-138, or 171-190 of SEQ ID NO: 1. In some embodiments, the assay may use two such tracer molecules, or all three.

[0061] Thus, the amount of long cTnT in a sample can be determined based on the extent of the binding reaction involving both the first binding molecule and the second binding molecule, with the aid of a previous measurement of a standard solution of known troponin concentration. In other words, quantifying long cTnT can involve the use of a calibration curve, i.e., a standard curve. The generation of a calibration curve is well known in the art and can be performed by plotting the binding reaction rate versus the concentration of troponin applied in a given standard solution. Any unknown concentration of long cTnT can then be determined by comparing the detected binding reaction rate with the corresponding rate achieved on the calibration curve at a known troponin concentration.

[0062] Thus, after quantifying the amount of long cTnT in a test sample, the measured amount of long cTnT can be compared to any appropriate reference amount of long cTnT (e.g., long cTnT concentrations from one or more apparently healthy subjects). In some embodiments, the reference amount is an amount from a population study, preferably conducted in a normal, apparently healthy population. A reference value from one or more apparently healthy subjects or from an apparently healthy population can be labeled a "normal reference amount." In selecting an appropriate reference amount, general considerations such as age range, gender, race, etc. can be considered. The comparison results can then be used for clinical decision making, such as identifying a patient suspected of having an acute MI as a patient who may actually have an acute MI or a patient who may not have an acute MI, and selecting an appropriate treatment procedure.

[0063] Surprisingly, when the assay included calculation of the ratio of long cTnT to standard cTnT measurable by a conventional high-sensitivity cTnT (hs-cTnT) assay (long / standard cTnT ratio) or the ratio of standard cTnT to long cTnT (standard / long cTnT ratio), the diagnostic performance of the assay was even more outstanding (AUC of 0.937 (CI95% 0.874–1.0) for long cTnT and 0.955 (CI95% 0.899–1.0) for the ratio of long cTnT to standard cTnT in differentiating NSTEMI from ESRD patients whose samples were collected within 24 hours of symptom onset). The traditional gold standard hs-cTnT assay commercially available from Roche Diagnostics uses a capture antibody that recognizes an epitope at amino acids 136-147 of SEQ ID NO: 1 and a tracer antibody conjugated to a detectable label that recognizes an epitope at amino acids 125-130 of SEQ ID NO: 1. Thus, the hs-cTnT assay targets the middle portion (i.e., the center portion) of the cTnT molecule, which detects all longer and shorter cTnT molecules that comprise this portion.

[0064] It is noteworthy that the assay of the present invention can, in some embodiments, utilize the hs-TnT assay of Roche Diagnostics or any other existing available equivalent hs-TnT assay to quantify the standard cTnT in the sample to be analyzed. However, the assay of the present invention is not limited to utilizing any available hs-TnT assay, such as the assay of Roche Diagnostics. Rather, in some embodiments, any one or more binding molecules that can specifically target and detect the central portion of human cTnT can be used in the present assay to quantify the standard cTnT.

[0065] Thus, the standard cTnT in the sample to be analyzed can be quantified by contacting the sample with a third binding molecule and a fourth binding molecule, both of which specifically bind to the region corresponding to amino acids 69-189 of SEQ ID NO: 1, preferably the region corresponding to amino acids 69-160 of SEQ ID NO: 1. Preferably, the third binding molecule is a capture molecule, such as a capture antibody, and the fourth binding molecule is a tracer molecule, such as a tracer antibody, or vice versa. In some embodiments, more than one capture molecule and / or tracer molecule can be used to improve detection. Typically, the number of capture and / or tracer molecules is 1 to 3, i.e., typically 1, 2 or 3.

[0066] In some embodiments, the third binding molecule and the fourth binding molecule are specific for different cTnT epitopes selected from the group consisting of epitopes corresponding to amino acids 67-86, amino acids 119-138, amino acids 125-130, amino acids 132-151, amino acids 136-147, amino acids 145-164, amino acids 171-190 of SEQ ID NO: 1.

[0067] As an alternative or in addition to quantifying standard cTnT, in some embodiments, the assay method may involve quantifying a third group of cTnT molecules, which includes all cTnT forms that include the C-terminal portion of cTnT (i.e., the region ranging from the C-terminal cleavage region to the C-terminal end). Such forms may be collectively referred to herein as "C-terminal cTnT". In some embodiments, the third group of cTnT molecules will be quantified with the aid of a fifth binding molecule and a sixth binding molecule that specifically bind to different epitopes in the region corresponding to amino acids 223-288 of SEQ ID NO: 1. Alternatively, the sixth binding molecule may be selected from the following binding molecules: a binding molecule that is capable of specifically binding to cardiac troponin I (cTnI; SEQ ID NO: 2), cardiac troponin C (cTnC; SEQ ID NO: 3), or an epitope formed when the cTnT form to be quantified is complexed with cTnI and cTnC or when cTnI and cTnC are complexed with each other. Some non-limiting examples of suitable cTnI epitopes are listed above. Preferably, the fifth binding molecule is a capture molecule, such as a capture antibody, and the sixth binding molecule is a tracer molecule, such as a tracer antibody, and vice versa. In some embodiments, more than one capture and / or tracer molecule can be used to improve detection. Typically, the number of capture and / or tracer molecules is 1 to 3, i.e., typically 1, 2 or 3.

[0068] As with the case of quantifying long cTnT (i.e., the first group of cTnT molecules), quantifying standard cTnT (i.e., the second group of cTnT molecules) or C-terminal cTnT (i.e., the third group of cTnT molecules) may involve utilizing an appropriate standard curve to determine the relative or absolute amount of standard cTnT or C-terminal cTnT in a sample.

[0069] The ratio of long cTnT to standard cTnT or the ratio of long cTnT to C-terminal cTnT can be calculated by simply dividing the measured amount of long cTnT by the measured amount of standard cTnT or C-terminal cTnT, respectively. The inverse ratio (by dividing the measured amount of standard cTnT or the measured amount of C-terminal cTnT by the measured amount of long cTnT) can also be used for calculation. The calculated ratio can then be compared to any corresponding appropriate reference ratio and used for clinical decision making, e.g., to identify patients whose samples have been analyzed using the present assay as those who may or may not have acute MI.

[0070] As in the case of an appropriate reference amount, a corresponding appropriate reference ratio (long / standard cTnT ratio, long / C-terminal cTnT ratio, standard / long cTnT ratio or C-terminal / long cTnT ratio, as the case may be) may be obtained from one or more apparently healthy subjects. In some embodiments, the reference ratio is a ratio from a population study, preferably conducted in a normal, apparently healthy population. A reference ratio from an apparently healthy subject or an apparently healthy population may be denoted as a "normal reference ratio". In selecting an appropriate reference ratio, general considerations such as age range, gender, race, etc. may be taken into account.

[0071] It should be understood that any appropriate reference amount or reference ratio (collectively referred to as reference value) can refer to an absolute value or a relative value. In addition, any reference value can be a range value. Therefore, the reference value to be used can be, for example, an absolute value or an absolute range value, or a relative value or a relative range value.

[0072] Typically, an increase in the measured long cTnT concentration or the long / standard cTnT ratio compared to the corresponding normal reference value or the corresponding normal reference range indicates the presence of M1. However, when the standard / long cTnT ratio is used, a decrease in the measured ratio compared to the corresponding normal reference ratio or the corresponding normal reference ratio range generally indicates the presence of MI. In some embodiments, a deviation (increase or decrease as described above) of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more of the measured long cTnT concentration, the long / standard cTnT ratio or the standard / long cTnT ratio compared to the corresponding normal reference value or range can be considered to indicate the presence of MI. Therefore, despite an elevation in standard cTnT, the absence of a significant deviation (no increase or decrease as described above) in the measured long cTnT concentration, long / standard cTnT ratio, or standard / long cTnT ratio compared to the corresponding normal reference value or range can be considered to indicate the absence of MI.

[0073] As an alternative or in addition to normal reference values ​​and / or normal reference ranges, the reference values ​​or reference range values ​​may, in some embodiments, be derived from one or more subjects with a known disease state (e.g., NSTEMI or STEMI) with a known delay time between symptom onset and blood sample collection, or with a known state of renal dysfunction.

[0074] In some embodiments, the reference value can be a value obtained from the same subject at an earlier time point. Such embodiments allow monitoring of any changes in a subject's long cTnT concentration, long / standard cTnT ratio, or standard / long cTnT ratio over time. Generally, the absence of significant changes over time indicates that the elevated standard cTnT level is due to a cause other than MI.

[0075] It is understood that calculated cTnT concentrations and ratios as well as reference concentrations and ratios may vary depending on various variables, such as the assay format, the type and amount of binding molecules used, the calibration of the assay, and the detection technology used to measure the concentration of the cTnT form in question.

[0076] The assay may be any available non-competitive assay type, as will be readily appreciated by those skilled in the art. Preferably, the assay is a non-competitive immunoassay. Non-limiting examples of suitable immunoassays include enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), counting immunoassay (CIA), fluorescent immunoassay (FIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), immuno-PCR assay, open sandwich immunoassay (OS), and microsphere-based immunoassay (MIA).

[0077] In some embodiments, the assay is a solid phase immunoassay, such as a lateral flow assay or a conventional sandwich assay performed on a solid surface (e.g., a microtiter plate or beads). In these assay formats, the capture molecule (i.e., the first or third binding molecule) is immobilized on the solid surface and the tracer molecule is detectably labeled. If the test sample contains the troponin species to be quantified, a detectable sandwich is formed between the troponin analyte and the capture molecule and the tracer molecule.

[0078] Non-limiting examples of suitable solid surfaces for solid phase assays include, but are not limited to, solid surfaces based on polystyrene, nitrocellulose or nylon membranes, glass or silicon. Any suitable technique available in the art can be used to fix the capture molecule on the solid surface, including but not limited to surface modification techniques, covalent coupling techniques and affinity fixation techniques. For example, biotin has a strong affinity for streptavidin. Therefore, when coated on a solid surface, streptavidin can be used as a fixative for biotinylated capture molecules (e.g., antibodies). Other affinity fixation techniques include, for example, DNA directional fixation, protein A and protein G mediated fixation, fixation by Fc binding peptides and aptamers, and fixation by material binding peptides or by metal affinity. Covalent coupling can be achieved by, for example, amines, carboxyls, thiols or carbohydrate groups in the targeted capture molecule or by using "click" chemistry.

[0079] The choice of detectable label to be conjugated or otherwise associated with the tracer molecule depends on the detection technology to be used, as will be readily understood by those skilled in the art. Suitable examples of detectable labels include, but are not limited to, optical agents, such as fluorescent labels, including various organic and / or inorganic small molecules, lanthanide chelates, upconversion nanoparticles and various fluorescent proteins and their derivatives, phosphorescent labels, chemiluminescent labels, electrochemiluminescent labels and chromogenic labels; radioactive labels, such as radionuclides that emit gamma rays, positrons, beta or alpha particles or X-rays; and enzymes, such as alkaline phosphatase (AP) or (horseradish) catalase (HRP); and oligonucleotides that form amplicons to be detected by PCR reactions. The association may be direct (e.g., by covalent bonds) or indirect (e.g., by a second binding agent, chelator or linker). Techniques for conjugating or otherwise associating detectable agents with tracer molecules (such as antibodies) are well known, for example, antibody labeling kits are commercially available from dozens of sources. Tracer molecules can also be expressed by recombinant technology as fusion proteins with detectable labels or detection tags.

[0080] In some embodiments, fluorescent labels are preferred, especially europium (III) chelates. Non-limiting examples of such chelates include [2,2',2",2'"-{[2-(4-isothiocyanatophenyl)ethylimino]bis-(methylene)bis{4-{[4-(α-galactopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis(methylenenitrile)}tetrakis(acetato)]europium(III) chelate.

[0081] Solid phase immunoassays can be either heterogeneous or homogeneous. In heterogeneous assays, any free analyte or tracer molecule must be physically separated from the assay reaction, for example by washing, whereas in homogeneous assays such separation is not required, making homogeneous assays more convenient.

[0082] In some preferred embodiments, the assays of the invention are heterogeneous solid phase immunoassays.

[0083] Homogeneous immunoassays can be performed not only in solid phase assay formats, but also in solutions. Such in-solution immunoassays are particularly advantageous because they do not require fixation or washing steps, making them simple and easy to perform. Examples of homogeneous assays include, but are not limited to, colorimetric assays, assays based on detection of ultraviolet signals, fluorescence resonance energy transfer (FRET) assays, proximity ligation immunoassays (PLA), proximity extension immunoassays (PEA), and luminescence assays based on oxygen channels (AlphaLISA and AlphaScreen). Nanomaterials, such as upconversion nanoparticles, noble metal nanoparticles, quantum dots (QD), and graphene oxide (GO) can also be used for homogeneous assays, as known in the art.

[0084] Some embodiments may employ proximity detection (PLA) technology to detect or quantify long cTnT and / or standard cTnT in a sample. Typically, this can be achieved by using different DNA oligonucleotides coupled to protein recognition molecules to form PLA probes. When the sample is contacted with the PLA probes, two PLA probes containing different DNA sequences will simultaneously bind to the same cTnT molecule to be detected or quantified. After the binding reaction, the DNA tails of the two probes are spatially close and hybridize to a third free oligonucleotide immediately after each other, which is complementary to the DNA sequence of the PLA probe. The 5' and 3' ends of the DNA tails of the two probes are connected by a ligase to form an amplicon to be quantified in a quantitative PCR reaction. The amount of connected single-stranded DNA is positively correlated with the amount of cTnT molecules to be measured in the sample.

[0085] Some embodiments may employ a competitive assay format, preferably a competitive immunoassay format, to quantify long cTnT. In such embodiments, only one binding molecule, preferably an antibody, is used that specifically binds to the C-terminal cleavage region of cTnT and whose binding to its epitope is severely impaired by cleavage of the cleavage region. By definition, since the C-terminal cleavage region of long cTnT is intact, such a binding molecule can correctly distinguish long cTnT from other forms of cTnT molecules (i.e., cTnT fragments cleaved at the C-terminal cleavage region) that may be present in a sample whose long cTnT content is to be quantified. For example, in some embodiments, long cTnT competes with a detectably labeled analyte analog (i.e., a detectably labeled cTnT molecule or a corresponding peptide comprising all or part of the intact C-terminal cleavage region) for binding to the binding molecule in a concentration-dependent manner. In other words, the higher the concentration of long cTnT in the sample, the less the detectably labeled analyte analog binds to the binding molecule, and thus the lower the signal. As is well known to those skilled in the art, in addition to the above examples, there are other competitive assay formats that can be used to quantify long cTnT. For example, the analyte analog does not need to be detectably labeled, especially if the analyte analog is immobilized on a solid surface and the binding molecule is detectably labeled.

[0086] The present invention also provides a kit for quantitatively testing the desired cTnT species in a sample and its use. The kit comprises: at least one first binding molecule, which can specifically bind to: a cTnT region corresponding to amino acids 201-288, preferably amino acids 223-288, of SEQ ID NO: 1, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed; and at least one second binding molecule, which can specifically bind to a cTnT region corresponding to amino acids 1-200, preferably amino acids 1-190, 69-200, 69-190, 164-200, or 164-190 of SEQ ID NO: 1. Alternatively, at least one first binding molecule is capable of specifically binding to the C-terminal cleavage region of cTnT, more specifically, to the region corresponding to amino acids 195-220 of SEQ ID NO:1, and at least one second binding molecule is capable of specifically binding to: the portion toward the N-terminus, more specifically, the region corresponding to amino acids 1-194, preferably amino acids 1-190, 69-194, 69-190, 164-194 or 164-190 of SEQ ID NO:1; the portion toward the C-terminus, more specifically, the region corresponding to amino acids 221-288, preferably amino acids 221-287, 224-287 or 224-288 of SEQ ID NO:1; cTnI (SEQ ID NO:2); cTnC (SEQ ID NO:3); or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed. Only long cTnT can be bound by both the first binding molecule and the second binding molecule. For solid phase applications, the first binding molecule is a capture molecule, preferably a capture antibody, and the second binding molecule is a tracer molecule, such as a tracer antibody, or vice versa. Non-limiting examples of suitable epitopes for the first binding molecule and the second binding molecule are listed above.

[0087] In some embodiments, the kit may include one, two or three tracer molecules, preferably antibodies, each tracer molecule specifically binding to a different epitope in the cTnT region corresponding to amino acids 1-200 of SEQ ID NO: 1. In some embodiments, the epitope comprises amino acids 67-86, amino acids 119-138, or amino acids 171-190 of SEQ ID NO: 1. In some embodiments, the first binding molecule specifically binds to amino acids 223-242 of SEQ ID NO: 1.

[0088] In some embodiments, the kit may include reagents for quantifying long cTnT using a competitive assay format. In such embodiments, the kit includes at least a seventh binding molecule that is capable of specifically binding to a cTnT region corresponding to amino acids 189-223 of SEQ ID NO: 1 or corresponding to amino acids 190-223 or 195-220 of SEQ ID NO: 1. In some embodiments, the kit further includes an analyte analog, i.e., a cTnT molecule or a peptide thereof, which includes at least a partially intact C-terminal cleavage region to compete with long cTnT for binding to the seventh binding molecule, preferably the seventh antibody. In some embodiments, the seventh binding molecule is immobilized on a solid surface, and the analyte analog is detectably labeled, or vice versa.

[0089] In some embodiments, the kit of the present invention may further comprise a binding molecule for quantifying standard cTnT. Therefore, the kit comprises a third binding molecule and a fourth binding molecule that can specifically bind to the cTnT region corresponding to amino acids 69-189 of SEQ ID NO: 1, preferably the region corresponding to amino acids 69-160 of SEQ ID NO: 1. All cTnT fragments containing the epitopes targeted by both the third binding molecule and the fourth binding molecule can be quantified together by the third binding molecule and the fourth binding molecule. For solid phase applications, one of the third binding molecule and the fourth binding molecule is a capture molecule, preferably a capture antibody, and the other binding molecule is a tracer molecule, such as a tracer antibody. Non-limiting examples of suitable epitopes for the third binding molecule and the fourth binding molecule are listed above. In some embodiments, the epitope targeted by the capture molecule comprises amino acids 136-147 of SEQ ID NO: 1, and the epitope targeted by the tracer molecule comprises amino acids 125-130 of SEQ ID NO: 1, and vice versa. In some embodiments, the kit of the present invention may further comprise a binding molecule for quantifying C-terminal cTnT. Therefore, the kit may include a fifth binding molecule and a sixth binding molecule that can specifically bind to the C-terminal region of cTnT corresponding to amino acids 223-288 of SEQ ID NO: 1. The whole cTnT and all cTnT fragments containing the epitopes targeted by both the fifth binding molecule and the sixth binding molecule can be quantified together by the fifth binding molecule and the sixth binding molecule. Alternatively, the sixth binding molecule can be a binding molecule that can specifically bind to cTnI, cTnC, or an epitope formed when the cTnT form to be quantified is complexed with cTnI and cTnC or when cTnC and cTnI are complexed, preferably an antibody. In this case, the whole cTnT and (i) all cTnT fragments containing the epitope targeted by the fifth binding molecule and (ii) all cTnT fragments complexed with cTnI and cTnC can be quantified together by the fifth binding molecule and the sixth binding molecule. For solid phase applications, one of the fifth binding molecule and the sixth binding molecule is a capture molecule, preferably a capture antibody, and the other binding molecule is a tracer molecule, such as a tracer antibody.

[0090] Antibodies suitable for use in the present invention are available in the art. Further antibodies can be developed by, for example, traditional animal immunization, or obtained from a recombinant expression library using phage display technology, including but not limited to phage display, ribosome display, bacterial cell surface display, yeast cell surface display or mammalian cell surface display.

[0091] The present assay and / or kit can be used for different purposes, such as for determining the possibility of acute MI in patients with acute MI symptoms. In other words, patients suspected of having acute MI can be identified as patients who may have acute MI or patients who may not have MI. Therefore, according to the test results, the present assay can help to exclude the possibility that the patient has MI or confirm the possibility that the patient has MI. For example, if the sample obtained from the patient does not show an increase in the amount of long cTnT compared to the normal reference value, or the ratio of long cTnT to standard cTnT is lower than the normal reference ratio or a predetermined cut-off limit, the patient suspected of having MI may not actually have acute MI. The experimental part demonstrates that long cTnT, in particular the long / standard cTnT ratio, is significantly increased in patients with acute MI, especially within 24 hours after MI.

[0092] It is also contemplated that the assay and / or kit may be used to distinguish patients with MI from patients who display elevated standard cTnT levels for reasons other than MI, including, for example, acute renal dysfunction, chronic kidney disease, ESRD, atrial fibrillation, and strenuous exercise.

[0093] In some embodiments, the present assays and / or kits can be used to identify patients who exhibit elevated standard cTnT levels and present with symptoms of acute MI as patients who actually have or may have acute MI, or as patients who do not or may not have acute MI despite symptoms. This assay provides a much-needed improved tool, particularly for emergency departments, as standard high-sensitivity cTnT assays lack adequate diagnostic performance for patients with transient or chronic elevations of high-sensitivity cardiac troponin.

[0094] In some embodiments, the assay method and / or kit can be used to distinguish patients with MI from patients with renal dysfunction (e.g., CKD or ESRD) when the patient shows an elevated standard cTnT. In fact, the experimental part shows that patients with chronic kidney disease, especially ESRD, only show very low long cTnT concentrations and very low long / standard cTnT ratios.

[0095] The above aspects and / or embodiments of the present invention can be expressed in different ways. It is noteworthy that, even without repetition, all the above alternative and / or preferred amino acid subsequences can also be applied to the embodiments described below.

[0096] At its simplest, the present invention provides an in vitro assay for detecting and / or quantifying long cTnT in a sample, the method comprising:

[0097] A)

[0098] i) contacting the sample with a first binding molecule and a second binding molecule, wherein the first binding molecule specifically binds to: a cTnT region corresponding to amino acids 201-288 of SEQ ID NO: 1, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed, and the second binding molecule specifically binds to a cTnT region corresponding to amino acids 1-200 of SEQ ID NO: 1; and

[0099] ii) detecting the presence of long cTnT in the sample if a specific binding reaction involving both the first binding molecule and the second binding molecule occurs, or quantifying long cTnT based on the binding reaction in step i), preferably with the aid of a calibration curve; or

[0100] B)

[0101] i) contacting the sample with a first binding molecule and a second binding molecule, wherein the first binding molecule specifically binds to a cTnT region corresponding to amino acids 195-220 of SEQ ID NO: 1, and the second binding molecule specifically binds to: a cTnT region corresponding to amino acids 1-194 of SEQ ID NO: 1, a cTnT region corresponding to amino acids 221-288, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed; and

[0102] ii) detecting the presence of long cTnT in the sample if a specific binding reaction involving both the first binding molecule and the second binding molecule occurs, or quantifying long cTnT based on the binding reaction in step i), preferably with the aid of a calibration curve.

[0103] The quantitative result obtained in step A or B can then be compared with an appropriate reference value, for example to determine whether the subject whose sample the long cTnT has been quantified is likely to have MI, thereby converting this method into an in vitro method for determining the likelihood of MI in a subject. According to the present invention, an increased amount of long cTnT compared to a normal reference value is indicative of MI.

[0104] Therefore, the above method can also be formulated as an in vitro assay for determining the likelihood of acute myocardial infarction (MI) in a subject, the method comprising:

[0105] i) measuring the amount of long cTnT in a sample obtained from the subject, the first group of cTnT molecules consisting of cTnT forms that are not cleaved in the region corresponding to amino acids 189-223 of SEQ ID NO: 1 and include a region corresponding to amino acids 205-215 of SEQ ID NO: 1; and

[0106] ii) determining the likelihood of acute MI in the subject based on said measurements.

[0107] In some embodiments, a method of determining the likelihood of acute MI in a subject comprises:

[0108] i) measuring the amount of long cTnT in the sample by contacting the sample with a first binding molecule that specifically binds to: a region of cTnT corresponding to amino acids 201-288 of SEQ ID NO: 1, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed, and a second binding molecule that specifically binds to a region of cTnT corresponding to amino acids 1-200 of SEQ ID NO: 1; or

[0109] i') measuring the amount of long cTnT in the sample by contacting the sample with a first binding molecule that specifically binds to a region of cTnT corresponding to amino acids 195-220 of SEQ ID NO: 1 and a second binding molecule that specifically binds to: a region of cTnT corresponding to amino acids 1-194 of SEQ ID NO: 1, a region of cTnT corresponding to amino acids 221-288, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed; and

[0110] ii) determining the likelihood of acute MI in the subject based on said measurements.

[0111] In some further embodiments, the method of determining the likelihood of acute MI in a subject comprises:

[0112] i) measuring the amount of long cTnT in the sample by contacting the sample with a binding molecule that specifically binds to the C-terminal cleavage region and an analyte analog (preferably cTnT or a peptide thereof comprising the entire C-terminal cleavage region or a portion thereof), wherein the long cTnT present in the sample competes with the analyte analog for binding to the binding molecule; and

[0113] ii) determining the likelihood of acute MI in the subject based on said measurements.

[0114] In some preferred assay formats, the analyte analog is detectably labeled.

[0115] Also provided is an in vitro method for calculating the ratio of long cTnT to standard cTnT in a sample. The method may include:

[0116] i) measuring the amount of long cTnT in the sample;

[0117] ii) measuring the amount of standard cTnT in the sample; and

[0118] iii) calculating the ratio of long cTnT to standard cTnT or the ratio of standard cTnT to long cTnT in the sample based on the measurements in steps i) and ii).

[0119] In some embodiments, a method of calculating the ratio of long cTnT to standard cTnT in a sample may include:

[0120] i) measuring the amount of long cTnT in the sample by contacting the sample with a first binding molecule that specifically binds to: a region of cTnT corresponding to amino acids 201-288 of SEQ ID NO: 1, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed, and a second binding molecule that specifically binds to a region of cTnT corresponding to amino acids 1-200 of SEQ ID NO: 1; or

[0121] ii) i') measuring the amount of long cTnT in the sample by contacting the sample with a first binding molecule that specifically binds to a region of cTnT corresponding to amino acids 195-220 of SEQ ID NO: 1 and a second binding molecule that specifically binds to: a region of cTnT corresponding to amino acids 1-194 of SEQ ID NO: 1, a region of cTnT corresponding to amino acids 221-288, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed; and measuring the amount of standard cTnT in the sample by contacting the sample with a third binding molecule and a fourth binding molecule that specifically bind to different epitopes in the region corresponding to amino acids 69-189 of SEQ ID NO: 1; and

[0122] iii) calculating the ratio of long cTnT to standard cTnT in the sample based on the measurements in steps i) or i') and ii).

[0123] The calculated ratio obtained in step iii) can then be used to determine, for example, whether a subject whose sample has been analyzed for the ratio of long cTnT to standard cTnT is likely to have MI, thereby converting this method into an in vitro method for determining the likelihood of MI in a subject. In some embodiments, the calculated ratio obtained in step iii) is compared to a corresponding reference ratio, preferably to a corresponding normal reference ratio for said determination.

[0124] It should be understood that the above method for calculating the ratio of long cTnT to standard cTnT can be converted into a method for calculating the ratio of standard cTnT to long cTnT by simply modifying step iii), so that the ratio of standard cTnT to long cTnT is calculated based on the measurements in steps i) or i') and ii), rather than calculating the ratio of long cTnT to standard cTnT. It is obvious to those skilled in the art that the above paragraph also applies to the method disclosed in this paragraph.

[0125] In some embodiments, the amount of standard cTnT can be measured using a commercially available conventional hs-cTnT assay.

[0126] In some embodiments, calculating the ratio of long cTnT to standard cTnT in a sample can be replaced by calculating the ratio of long cTnT to C-terminal cTnT. Therefore, an in vitro method for calculating the ratio of long cTnT to C-terminal cTnT in a sample is also provided, the method comprising:

[0127] i) measuring the amount of long cTnT in the sample;

[0128] ii) measuring the amount of C-terminal cTnT in the sample; and

[0129] iii) calculating the ratio of long cTnT to C-terminal cTnT or the ratio of C-terminal cTnT to long cTnT in the sample based on the measurements in steps i) and ii).

[0130] More specifically, the method of calculating the ratio of long cTnT and C-terminal cTnT in a sample may include:

[0131] i) measuring the amount of long cTnT in the sample by contacting the sample with a first binding molecule that specifically binds to: a region of cTnT corresponding to amino acids 201-288 of SEQ ID NO: 1, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed, and a second binding molecule that specifically binds to a region of cTnT corresponding to amino acids 1-200 of SEQ ID NO: 1; or

[0132] i') measuring the amount of long cTnT in the sample by contacting the sample with a first binding molecule that specifically binds to a region of cTnT corresponding to amino acids 195-220 of SEQ ID NO: 1 and a second binding molecule that specifically binds to: a region of cTnT corresponding to amino acids 1-194 of SEQ ID NO: 1 or a region of cTnT corresponding to amino acids 221-288, cTnI, cTnC, or an epitope formed when long cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed; and

[0133] ii) measuring the amount of C-terminal cTnT in the sample by contacting the sample with a fifth binding molecule that specifically binds to an epitope in the region corresponding to amino acids 223-288 of SEQ ID NO: 1 and a sixth binding molecule that specifically binds to: a different epitope in the region corresponding to amino acids 223-288 of SEQ ID NO: 1, cTnI, cTnC, or an epitope formed when C-terminal cTnT is complexed with cTnI and cTnC or when cTnI and cTnC are complexed; and

[0134] iii) calculating the ratio of long cTnT to C-terminal cTnT or the ratio of C-terminal cTnT to long cTnT in the sample based on the measurements in steps i) or i') and ii).

[0135] In some embodiments, the first binding molecule and / or the sixth binding molecule is capable of specifically binding to: cTnI comprising the amino acid sequence shown in SEQ ID NO:2, preferably the amino acids corresponding to amino acids 23-197 in SEQ ID NO:2, cTnC comprising the amino acid sequence shown in SEQ ID NO:3, or an epitope formed when the cTnT form to be quantified is complexed with cTnI and cTnC or when cTnI and cTnC are complexed.

[0136] Likewise, the calculated ratio obtained in step iii) can be used to determine whether a subject, for example, whose sample has been analyzed for the ratio of long cTnT to C-terminal cTnT, is likely to suffer from MI, thereby converting the method into an in vitro method for determining the likelihood of MI in a subject.

[0137] Quantifying long cTnT or calculating the ratio of long cTnT to standard cTnT or the ratio of long cTnT to C-terminal cTnT can provide substantial assistance for clinical decision making to select necessary follow-up tests and appropriate treatment procedures. In some embodiments, the present method of determining the likelihood that a subject has MI can also include therapeutic intervention. Once it is determined that a subject may have an acute MI, he / she can undergo, for example, coronary angiography, followed by revascularization and administration of antithrombotic therapy if necessary.

[0138] Thus, a method of treating MI in a subject in need thereof is also provided, wherein the method first comprises determining the likelihood that a subject suspected of having MI actually has MI, and if the subject is determined or identified as likely to have MI, subjecting the subject to any appropriate MI treatment, such as coronary revascularization and administration of antithrombotic therapy.

[0139] Example 1. Exemplary Three-Tracer Long cTnT Assay

[0140] method

[0141] patient

[0142] In the Troponin Fragments in Myocardial Injury (Tropo-Fragm) study (ClinicalTrials.gov Identifier: NCT04465591), we evaluated cTnT fragments in 46 patients with non-ST-segment elevation myocardial infarction (NSTEMI), 71 patients with ST-segment elevation myocardial infarction (STEMI), and 40 patients with ESRD receiving maintenance hemodialysis. The clinical characteristics of the study groups are shown in Table 1. Coronary angiography was performed in 44 patients (95.7%) in the NSTEMI group, 36 patients (78.3%) were treated with percutaneous coronary intervention (PCI), and 6 patients (13%) underwent coronary artery bypass grafting. All patients in the STEMI group underwent coronary angiography and primary PCI. All participants provided written informed consent. The study conformed to the Declaration of Helsinki as revised in 2002, and the study protocol was approved by the Medical Ethics Committee of the Hospital District of Southwest Finland.

[0143] Table 1. Baseline characteristics of patients in the study groups.

[0144]

[0145] Data are presented as cases / number (percentage or median (IQR)). ESRD denotes end-stage renal disease; NSTEMI, non-ST-segment elevation myocardial infarction; STEMI, ST-segment elevation myocardial infarction, IQR = interquartile range

[0146] Blood sampling

[0147] For patients with MI, blood samples were collected as soon as possible after admission. For patients with ESRD, blood samples were collected in the dialysis clinic before and after dialysis. The certified laboratory service of Turku University Hospital (TYKSLAB) was responsible for processing blood samples. Immediately upon arrival at the laboratory, 5 ml lithium heparin tubes were centrifuged at 2200 g for 15 min, and plasma was separated and stored at –70 °C until analysis. Samples were thawed at room temperature, mixed to ensure homogeneity, and centrifuged before analysis.

[0148] Laboratory methods

[0149] Standard cTnT assay. All plasma samples were analyzed using the Cobas 8000 system (e801 module) (Roche Diagnostics GmbH, Mannheim, Germany) using the Elecsys Troponin Ths kit. The Elecsys Troponin Ths assay uses two monoclonal antibodies that specifically target the central portion of human cardiac troponin T. The amino acid residue (aar) numbering in this article is based on the amino acid sequence of troponin T isoform 6 (also known as TnT3 (UniProtKB / Swiss-Prot entry P45379-6), the major isoform of troponin in the adult heart. 16 The capture antibody of the standard cTnT assay recognizes the epitope at aar 136-147, and the tracer antibody conjugated to tris(2,2'-bipyridyl)ruthenium(II) complex (Ru(bpy)) recognizes the epitope at aar 125-130. Therefore, the assay targets the middle part of the cTnT molecule and detects all longer and shorter cTnT molecules that include this part. The assay uses electrochemiluminescence detection. The calibration of the Roche assay is based on the Elecsys Troponin T STAT assay, which was originally standardized to the Enzymun-Test Troponin T method. The laboratory reporting limit for the Roche assay is 5 ng / l.

[0150] Long cTnT assay design. A novel sensitive time-resolved immunofluorescence assay was developed for the detection of long cTnT and the measurement of troponin fragments using the ratio of long cTnT to standard cTnT (cTnT ratio). The long cTnT assay adopts a sandwich immunoassay format and utilizes time-resolved fluorescence (TRF) as the measurement platform. The assay is based on the use of a capture antibody targeting the C-terminal portion of cTnT (mab 7E7, aar 223-242) and a tracer antibody combination targeting the portion towards the N-terminus of cTnT (mab 7G7, aar 67-86 in SEQ ID NO:1; mab 329cc, aar 119-138 in SEQ ID NO:1; mab 1C11cc, aar 171-190 in SEQ ID NO:1) ( Figure 1 ). The selected antibody combination excludes most fragment forms of cTnT, thereby facilitating the detection of long cTnT molecules spanning multiple cleavage sites located at aar 190-233. The use of multiple tracer antibodies with different epitopes can enhance the signal of a single cTnT molecule.

[0151] Monoclonal antibodies (mabs) for long cTnT assays were from Hytest Ltd (Turku, Finland). Human cardiac troponin ITC complex (Hytest Ltd, Turku, Finland) was used as a calibrator in the immunoassay, diluted in TSA-BSA buffer (tris-HCl [50 mmol / l] pH 7.75; NaCl [150 mmol / l]; NaN3 [0.5 g / l]; bovine serum albumin [75 g / l, Probumin, Merck, Darmstadt, Germany]) and stored at -20°C until use.

[0152] Antibodies were conjugated to biotin or europium chelate for long cTnT assays. The capture antibody (7E7) was conjugated to a 30-fold molar excess of biotin isothiocyanate (Department of Biotechnology, University of Turku, Turku, Finland) in sodium carbonate-bicarbonate buffer (50 mmol / l; pH 9.8). The reaction mixture was left at room temperature for 4 h, and then the buffer was exchanged to TSA buffer [50 mmol / l] pH 7.75; NaCl [150 mmol / l]; NaN3 [0.5 g / l] using NAP-10 and PD-10 columns (GE Healthcare). The mAb concentration of the collected eluate was measured at 280 nm using a UV-VIS spectrophotometer. 1 g / l of DTPA-purified BSA (Perkin Elmer) was added to the mAb preparation, and the solution was then filtered through a 0.22 μm pore size filter and stored at 4°C.

[0153] The tracer antibodies (7G7, 329cc, and 1C11cc) were conjugated with a 30- to 35-fold molar excess of intrinsically fluorescent [2,2',2",2",2"'-{[2-(4-isothiocyanatophenyl)ethylimino]bis-(methylene)bis{4-{[4-(α-galactopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis(methylenenitrile)}tetra(acetic acid)]europium(III) chelate 17 (Department of Biotechnology, University of Turku, Turku, Finland). The conjugation reaction was performed in sodium carbonate-bicarbonate buffer (50 mmol / l; pH 9.8) at room temperature overnight (18-20 h). The labeled antibodies were purified using an FPLC system equipped with a Superdex 200HR 10 / 30 gel filtration column (TSA buffer as eluent). Fractions containing labeled conjugated mAb were identified using UV detector and TRF measurement (Arcus 1230, Wallac Oy). Selected fractions containing labeled mAb were pooled and 1 g / l DTPA-purified BSA was added, then the solution was filtered through a 0.22 μm pore size filter and stored at 4°C.

[0154] Long cTnT assay protocol. 200 ng of biotinylated capture antibody in 25 μl buffer solution RED (Kaivogen) was added to streptavidin-coated microtiter plate wells (Kaivogen) and incubated at room temperature for 1 hour. After incubation, the microtiter plate wells were washed with wash buffer (Kaivogen). Tracer antibodies (100 ng each) in 40 μl tracer buffer (Tris-HCl [100 mmol / l] pH 7.75; NaCl [600 mmol / l]; NaN3 [0.5 g / l]; BSA [25 g / l]; casein [4 g / l]; bovine-γ-globulin [0.6 g / l]; native mouse IgG [0.8 g / l]; denatured mouse IgG [0.05 g / l]) were added to the wells. Calibrator or sample (30 μ L per hole) is added to the hole, set three replicates, and cover the hole with sealing tape and incubate 1 hour (iEMS incubator / oscillator, Thermo Electron Corporation / Labsystems) at 900rpm at +36 ℃.Then wash the hole with washing buffer and dry 5 minutes in hot air stream.After the plate is cooled to room temperature, measure TRF signal (VictorX4, PerkinElmer), excitation wavelength is 340nm, emission wavelength is 615nm, measurement delay is 250μs, and measurement window is 750μs.All analyses are repeated three times.

[0155] Analytical performance of the long cTnT assay. The analytical detection limit of the long cTnT assay (blank mean + 3SD) was determined to be 10.0 ng / L, and the accuracy target of 20% CV was achieved at 13.8 ng / L. The assay had good linearity with STEMI plasma diluted in TSA-BSA buffer, and the ITC calibrants spiked into plasma samples were adequately recovered.

[0156] Statistical analysis

[0157] Continuous variables were reported as mean ± standard deviation (SD) and median (interquartile range [IQR]) for normally distributed and skewed variables, respectively. Normality of continuous covariates was analyzed using the Shapiro-Wilk test and the Kolmogorov-Smirnov test. Unpaired t-test or one-way analysis of variance and Mann-Whitney test or Kruskal-Wallis test were used to compare normally distributed and skewed continuous variables, respectively. In the analysis between multiple subgroups, pairwise comparisons were performed with the Bonferroni correction. Categorical covariates among study subgroups were compared using Pearson's χ2 or Fisher's exact test. Categorical variables were expressed as absolute and relative (percentage) frequencies. Linear regression models were used to test the association between continuous independent variables and dependent variables. Standard cTnT and long cTnT levels before and after dialysis in the ESRD group were compared using the Wilcoxon signed-rank test.

[0158] Receiver operating characteristic (ROC) curve analysis was performed to estimate the area under the curve (AUC) to measure the discriminatory ability of long cTnT and cTnT ratio between the ESRD group and the NSTEMI group. All tests were two-sided and significance was set at p < 0.05. All analyses were performed using IBM SPSS Statistics software version 26.0.

[0159] result

[0160] Long cTnT, standard cTnT, and long / standard cTnT ratio in STEMI, NSTEMI, and ESRD groups

[0161] STEMI patients had the highest standardized cTnT concentrations (Table 2). All ESRD patients except one had standardized cTnT values ​​above the uniform 99th percentile of 14 ng / L, but the standardized cTnT concentrations were not significantly different from those in the NSTEMI group (76 [50-124] ng / L vs. 141 [62-312] ng / L, p = 0.063) (Table 2, Figure 2). The long cTnT concentration in ESRD patients was lower than that in NSTEMI and STEMI patients (3 [1-6] ng / L vs. 30 [10-186] ng / L vs. 442 [154-1235] ng / L, p < 0.001 for both comparisons) (Table 2). Therefore, the ratio of long cTnT to standard cTnT in ESRD patients was lower than that in NSTEMI and STEMI patients (0.04 [0.01-0.08] vs. 0.20 [0.13-0.36] vs. 0.38 [0.17-0.66], p < 0.001 for both comparisons) (Table 2, Figure 2 and Figure 3 ).

[0162] Table 2. Renal function, standard and long cTnT and their ratios in the study groups.

[0163]

[0164] Data are presented as median and interquartile range (IQR).

[0165] ESRD denotes end-stage renal disease; NSTEMI, non-ST-segment elevation myocardial infarction; STEMI, ST-segment elevation myocardial infarction; eGFR, estimated glomerular filtration range; cTnT, cardiac troponin T.

[0166] Changes in the long / standard cTnT ratio after myocardial infarction

[0167] A delay between symptom onset and study sample could be determined in 109 (93%) patients with myocardial infarction. The long / standard cTnT ratio decreased over time with a mean half-life of 13.9 hours. Figure 4 Thus, patients with a delay of ≤24 h (n=23 (53.5%) and n=32 (48.5%) in NSTEMI and STEMI patients, respectively) had higher cTnT ratios than those with longer delays, both in the NSTEMI group (median 0.29 (0.20-0.72) [delay ≤24 h] vs. 0.15 (0.10-0.20) [delay >24 h], p<0.001) and the STEMI group (median 0.62 (0.41-0.93) [delay ≤24 h] vs. 0.22 (0.11-0.38) [delay >24 h], p<0.001).

[0168] Long / standard cTnT ratio and hemodialysis

[0169] In univariate linear regression analysis, baseline eGFR was not associated with the cTnT ratio (β 0.300, CI 95% 0.0-0.013, p = 0.053) in the NSTEMI group or the STEMI group (β 0.045, CI 95% -0.004-0.006, p = 0.714). In ESRD patients, hemodialysis had no significant effect on long cTnT levels but resulted in a slight decrease in standard cTnT concentrations and a slight increase in the long / standard cTnT ratio (0.04 vs 0.06, p = 0.002) ( Figure 5 ).

[0170] Diagnostic performance of the long / standard cTnT ratio

[0171] In ROC curve analysis, the cTnT ratio showed excellent predictive ability in distinguishing ESRD from NSTEMI patients, with AUCs of 0.906 (CI95% 0.837-0.975) (including all NSTEMI patients) and 0.955 (CI95% 0.899-1.0) (including only NSTEMI patients with a delay of ≤24 hours between symptom onset and study sample collection). The cTnT ratio was significantly superior to standard cTnT in distinguishing ESRD from NSTEMI (standard cTnT AUC 0.688 (CI95% 0.575-0.800) for all NSTEMI patients and 0.609 (CI95% 0.454-0.764) for NTSEMI patients with a delay of <24 hours between symptom onset and sample collection). The optimal cTnT ratio cutoff of 0.105 had a sensitivity of 84.4% and a specificity of 86.8% in the ROC analysis for distinguishing ESRD patients from NSTEMI patients (including all patients). When only NSTEMI patients with a delay of ≤24 hours between symptom onset and blood sample collection were included, the sensitivity was 91.3% and the specificity was 94.7% with a cutoff of 0.145 ( Figure 6 ).

[0172] Example 2. Exemplary single tracer long cTnT assay

[0173] Materials and methods

[0174] Long cTnT assay using a single tracer

[0175] The capture antibody (Hytest antibody mab 7E7, aar 223-242) which had been biotinylated as described in Example 1 was added to the streptavidin-coated microtiter plate wells (Kaivogen) at 200 ng in 25 μl buffer solution RED (Kaivogen) and incubated for 1 hour at room temperature. After incubation, the microtiter plate wells were washed with wash buffer (Kaivogen). A tracer antibody (Hytest antibody mab 7G7, aar 67-86; mab 329cc, aar 119-138; or mab1C11cc, aar 171-190, labeled with an intrinsically fluorescent europium (III) chelate as described in Example 1) was added to the wells at 100 ng in 10 μl of tracer buffer (Tris-HCl [100 mmol / l] pH 7.75; NaCl [600 mmol / l]; NaN3 [0.5 g / l]; BSA [25 g / l]; casein [4 g / l]; bovine-γ-globulin [0.6 g / l]; native mouse IgG [0.8 g / l]; denatured mouse IgG [0.05 g / l]). Calibrator or sample (30 μ L per hole) is added to the hole, three replicates are set, and the hole is covered with sealing tape, and incubated for 1 hour (iEMS incubator / oscillator, Thermo Electron Corporation / Labsystems) at 900 rpm at +36 ℃. Then wash the hole with washing buffer and dry it in a hot air stream for 5 minutes. After the plate is cooled to room temperature, TRF signal (VictorX4, PerkinElmer) is measured, and the excitation wavelength is 340nm, and the emission wavelength is 615nm, and the measurement delay is 250μs, and the measurement window is 750μs. All analyses are repeated three times.

[0176] Standard cTnT assay

[0177] Samples were analyzed using a standard cTnT assay similar to Example 1

[0178] Patients, samples, and calibrators

[0179] This study included a subset of lithium heparin samples described in Example 1 collected from the patients described in Example 1. Similar calibrators as in Example 1 were used in this study.

[0180] Statistical analysis

[0181] Continuous variables are reported as medians (interquartile range [IQR]) due to uneven distribution and were compared using the Mann-Whitney test. All analyses were performed using IBM SPSS Statistics software, version 26.0.

[0182] result

[0183] Using the long cTnT assay using only mab 329 as the tracer antibody, long cTnT concentrations were significantly higher in samples collected from 10 consecutive NSTEMI patients within 24 hours of symptom onset than in samples from 40 ESRD patients (69 ng / L [25-306 ng / L] vs. 10 ng / L [6-13 ng / L], p < 0.001). Standard cTnT concentrations overlapped significantly in the same group (132 ng / L [95-276 ng / L] vs. 76 ng / L [50-122 ng / L], p = 0.450). In addition, the ratio of long cTnT to standard cTnT was also significantly higher in the same NSTEMI patients (0.50 [0.28-0.80] vs. 0.12 [0.07-0.20], p < 0.001) ( Figure 7 ).

[0184] The long cTnT assay using mab 7G7 or mab 1C11 as a single tracer antibody measured similar high concentrations in samples collected from STEMI patients as the long cTnT assay using mab 329 alone as a tracer or the assay of Example 1 using a combination of mab 329, mab 1C11, and mab7G7 as tracers.

[0185] Example 3. Exemplary long cTnT assay using capture antibodies targeting cTnI or cTnI-TnC complex

[0186] Materials and methods

[0187] Long cTnT assay using capture antibodies that bind to cTnI or cTnI-TnC complex

[0188] cTnI targeting capture antibodies (anti-cTnI mab 19C7, aar 41-49, HyTest and 8I7, aar 137-148, International Point of Care inc) and cTnI-TnC complex targeting antibodies (Tcom8, epitope present only when cTnI and cTnC are complexed, Hytest) were biotinylated in a similar manner as described for the capture antibodies in Example 1. Antibodies (100 ng 19C7 and 100 ng 8I7 or 200 ng Tcom8) in 25 μl buffer solution RED (Kaivogen) were added to streptavidin-coated wells and incubated for 1 hour at room temperature. After incubation, the microtiter wells were washed with wash buffer (Kaivogen). A combination of cTnT-targeting tracer antibodies (Hytest antibodies mab 7G7, aar 67-86; mab 329cc, aar 119-138; and mab 1C11cc, aar 171-190, labeled with an intrinsically fluorescent europium (III) chelate as described in Example 1) was added to the wells at 100 ng each in 40 μl of tracer buffer (Tris-HCl [100 mmol / l] pH 7.75; NaCl [600 mmol / l]; NaN3 [0.5 g / l]; BSA [25 g / l]; bovine-γ-globulin [0.6 g / l]; native mouse IgG [0.8 g / l]; denatured mouse IgG [0.05 g / l]; casein [4 g / l]). Calibrator or sample (30 μ L per well) is added to wells, three replicates are set, and wells are covered with sealing tape, incubated at +36 ° C for 1 hour with 900 rpm (iEMS incubator / oscillator, ThermoElectron Corporation / Labsystems). Wells are then washed six times with wash buffer and dried in a hot air stream for 5 minutes. After the plate is cooled to room temperature, TRF signals (VictorX4, PerkinElmer) are measured, with an excitation wavelength of 340 nm, an emission wavelength of 615 nm, a measurement delay of 250 μ s, and a measurement window of 750 μ s.

[0189] Other assays

[0190] Samples were analyzed using a standard cTnT assay (ie, the commercial Roche hs-cTnT assay) similar to Example 1. Samples were also analyzed using the long cTnT assay described in Example 1, wherein the capture antibody was anti-cTnT mab 7E7 (Hytest).

[0191] Patients, samples, and calibrators

[0192] This study included a small subset of lithium heparin plasma samples described in Example 1 collected from the patients described in Example 1. This study used a similar calibrator as in Example 1 (Troponin ITC Complex from Hytest).

[0193] result

[0194] All assays (standard cTnT assay, long cTnT assay using anti-cTnT capture mab, long cTnT assay using anti-cTnI capture mab, and long cTnT assay using anti-cTnI-TnC complex capture mab) produced substantially elevated results in STEMI patients (Table 1). While the standard cTnT assay also provided substantially elevated results in ESRD patients, the other assays detected nothing or only negligible amounts in ESRD samples (Table 1).

[0195] The different values ​​detected by different long cTnT assays for STEMI patients may result from different fractions of cTn complexes in endogenous samples and the calibration materials used in the studies (e.g., perhaps due to instability, the amount of complexed cTn in the calibration material is lower, which would lead to increased endogenous sample results for assays using complex-dependent epitopes). However, the results show that it is possible to distinguish between MI and ESRD patients using each of the described long cTnT assay settings.

[0196] Table 3. Samples from STEMI and ESRD patients analyzed using the long cTnT assay with different settings.

[0197]

[0198] Example 4. Exemplary long cTnT assay using upconverting nanoparticles as labels

[0199] Materials and methods

[0200] Conjugation of upconversion nanoparticles to antibodies

[0201] Oleic acid-capped NaYF4:17% Yb 3+ , 3%Er 3+Upconverting nanoparticles (UCNPs) (University of Turku, Finland) were coated with polyacrylic acid (PAA) and conjugated with a monoclonal anti-cTnT tracer antibody (Hytest) as previously described (Raiko et al., Clin Chim Acta. 2021 Dec; 523: 380-385) using 2.5 mM EDC (mab 1C11, aar 171-190) or 30 mM EDC (mab 329cc, aar 119-138 and mab 7G7, aar 67-86).

[0202] Long cTnT assay using upconversion nanoparticles

[0203] Anti-cTnT antibody 7E7 (aar 223-242, HyTest) used as capture antibody was biotinylated as in Example 1. 30 minutes before the start of the assay, a tracer dilution containing 4 ng / μl of one UCNP-conjugated tracer antibody (mab 329cc, mab 1C11, or mab 7G7) was prepared using assay buffer (Kaivogen) supplemented with 1 mM KF, 0.05% PAA, 0.2% skim milk powder, 0.08% native mouse IgG, and 0.005% denatured mouse IgG. 200 ng of capture antibody in 50 μl of assay buffer was added to the wells of a C8 Lockwell LUMI White Maxisorp microtiter plate (Thermo Scientific), which had been coated with 1 μg / well streptavidin (Biospa) as previously described (Raiko et al., Clin ChimActa. 2021 Dec; 523: 380-385). After incubation for 30 minutes at room temperature with slow shaking, the wells were washed with wash buffer (Kaivogen).

[0204] Calibrators and samples were diluted in sample buffer (37.5 mM Tris pH 8, 500 mM NaCl, 0.06% bovine gamma globulin, 2.5% BSA, 5% D-trehalose, 37.5 U / ml heparin, 0.08% native mouse IgG, 0.005% denatured mouse IgG, 0.2% casein, 0.0375% NaN3) at a sample to buffer ratio of 4: 1. A volume of 50 μl of sample or calibrator was added to the wells. After incubation at room temperature for 30 minutes with slow shaking, the wells were washed with wash buffer.

[0205] The tracer dilutions were sonicated and 50 μl of the dilutions were added to each well. After incubation at room temperature for 15 minutes with slow shaking, the wells were washed with wash buffer and allowed to dry for 1.5 hours before signal measurement. Upconversion luminescence was measured from the bottom of the wells at 540 nm using a modified Plate Chameleon microplate reader (Hidex) equipped with 980 nm laser excitation.

[0206] Patients, samples, and calibrators

[0207] This study also included the two STEMI patients included in Example 1. Lithium heparin plasma samples and serum samples were collected from the patients at the same time. This study used a similar calibrator as in Example 1 (Troponin ITC complex from Hytest).

[0208] result

[0209] Using the heparin plasma samples of both patients, all assays produced significantly elevated results (mean 420 ng / L, SD 141 ng / L). However, using the serum samples, only the assays using 1C11 or 329cc as tracer antibodies produced significantly elevated results, showing an average of 75% (SD 3%) of the levels detected using the same assays in the heparin plasma samples. When using the tracer antibody 7G7, the results for the serum samples were significantly lower than the heparin plasma samples. Compared to the concentrations detected in the heparin plasma samples using the same assay, the concentrations detected in the serum using the 7G7 tracer antibody were only 6% of those detected in patient 1 and 8% of those detected in patient 2.

[0210] The epitope targeted by antibody 7G7 is located at aar 67-86. There is a prominent cTnT cleavage site between aar 68 and 69. Thrombin is known to cleave cTnT between aar 68 and 69. Since the coagulation pathway is activated when serum samples are prepared, thrombin is also activated when serum samples are generated. Therefore, in serum samples, cTnT is cleaved between aar 68 and 69 and the epitope targeted by antibody 7G7 is destroyed. As a result, we see low concentrations of long cTnT in serum samples utilizing mab 7G7 as a tracer antibody. This effect was not observed with tracer antibodies 1C11 and 329cc because they target epitopes closer to the C-terminal end of cTnT.

[0211] These results highlight that the vast majority of cTnT in the circulation of MI patients is in a form that is not truncated from the N-terminus, and therefore it is also important to measure the intact form of cTnT.

[0212] For those skilled in the art, as technology advances, the basic idea of ​​the invention can be implemented in various ways. Therefore, the present invention and its embodiments are not limited to the above-described embodiments, but can be varied within the scope of the claims.

Claims

1. An in vitro assay for determining the likelihood of acute myocardial infarction (MI) in a subject, the method comprising: i) measuring the amount of a first group of cardiac troponin (cTnT) molecules in a sample obtained from the subject, the first group of cTnT molecules consisting of intact cTnT and a C-terminal cTnT form that is not cleaved in the region corresponding to amino acids 189-223 of SEQ ID NO: 1 and comprises a region corresponding to amino acids 205-215 of SEQ ID NO: 1; and ii) determining the likelihood of acute MI in said subject based on said measurements.

2. The in vitro assay method according to claim 1, wherein the amount of the first group of cTnT molecules in the sample is measured using a first binding molecule and a second binding molecule, wherein the first binding molecule specifically binds to the cTnT region corresponding to amino acids 201-288 of SEQ ID NO: 1, cardiac troponin I (cTnI), cardiac troponin C (cTnC), or an epitope formed when the first group of cTnT molecules are complexed with cTnI and cTnC or when cTnI and cTnC are complexed, and the second binding molecule specifically binds to the cTnT region corresponding to amino acids 1-200 of SEQ ID NO:

1.

3. The in vitro assay method according to claim 2, wherein the first binding molecule is a capture molecule immobilized on a solid surface and the second binding molecule is a tracer molecule comprising a detectable label. 4 . The in vitro assay method according to claim 3 , wherein the capture molecule specifically binds to the cTnT epitope corresponding to amino acids 223-242 of SEQ ID NO:

1.

5. The in vitro assay according to claim 3 or 4, wherein one, two or three tracer molecules are used, each tracer molecule specifically binding to a different cTnT epitope selected from the epitopes corresponding to amino acids 67-86, 119-138 or 171-190 of SEQ ID NO:

1.

6. The in vitro assay method according to claim 1, wherein the amount of the first group of cTnT molecules is determined by a competitive assay and the amount of the first group of cTnT molecules in the sample is measured using a binding molecule that specifically binds to the cTnT region corresponding to amino acids 190-223, preferably amino acids 195-220 of SEQ ID NO:

1.

7. The in vitro assay method according to claim 1, wherein the amount of the first group of cTnT molecules in the sample is measured using a first binding molecule and a second binding molecule, wherein the first binding molecule specifically binds to the cTnT region corresponding to amino acids 195-220 of SEQ ID NO: 1, and the second binding molecule specifically binds to: the cTnT region corresponding to amino acids 1-194 of SEQ ID NO: 1, the cTnT region corresponding to amino acids 221-288, cTnI, cTnC, an epitope formed when the C-terminal cTnT is complexed with cTnI and cTnC, or an epitope formed when cTnI and cTnC are complexed.

8. The in vitro assay method according to claim 7, wherein the first binding molecule is a capture molecule immobilized on a solid support and the second binding molecule is a tracer molecule comprising a detectable label, or vice versa.

9. The in vitro assay according to any one of claims 1 to 8, further comprising: - measuring the amount of a second group of cTnT molecules in said sample, said second group of cTnT molecules consisting of all cTnT molecules in said sample comprising a cTnT central region; or - measuring the amount of a third group of cTnT molecules in the sample, the third group of cTnT molecules consisting of all cTnT molecules in the sample comprising a cTnT terminal region; and - calculating a ratio of the first group of cTnT molecules to the second group of cTnT molecules, a ratio of the second group of cTnT molecules to the first group of cTnT molecules, a ratio of the first group of cTnT molecules to the third group of cTnT molecules, or a ratio of the third group of cTnT molecules to the first group of cTnT molecules; and - determining the likelihood of acute MI in said subject based on the calculated ratio.

10. The in vitro assay method according to claim 9, wherein the amount of the second group of cTnT molecules in the sample is measured using a third binding molecule and a fourth binding molecule, which specifically bind to different epitopes in the region corresponding to amino acids 69-189 of SEQ ID NO:

1.

11. The in vitro assay method according to claim 10, wherein the third binding molecule specifically binds to the epitope corresponding to amino acids 136-147 of SEQ ID NO: 1, and the third binding molecule is preferably a capture molecule immobilized on a solid surface.

12. The in vitro assay method according to claim 10 or 11, wherein the fourth binding molecule specifically binds to an epitope corresponding to amino acids 125-130 of SEQ ID NO: 1, and the fourth binding molecule is preferably a tracer molecule comprising a detectable label.

13. The in vitro assay method according to claim 9, wherein the amount of the third group of cTnT molecules in the sample is measured using a fifth binding molecule and a sixth binding molecule, the fifth binding molecule and the sixth binding molecule specifically binding to different epitopes in the region corresponding to amino acids 223-288 of SEQ ID NO:

1.

14. The in vitro assay method according to claim 9, wherein the amount of the third group of cTnT molecules in the sample is measured using a fifth binding molecule and a sixth binding molecule, the fifth binding molecule specifically binding to an epitope in the region corresponding to amino acids 223-288 of SEQ ID NO: 1, and the sixth binding molecule specifically binding to cTnI, cTnC, or an epitope formed when the third group of cTnT molecules are complexed with cTnI and cTnC or when cTnI and cTnC are complexed.

15. The in vitro assay according to any one of claims 1 to 14, for excluding the possibility that a subject suffers from MI; confirming the possibility that a subject suffers from MI; distinguishing patients suffering from MI from patients showing elevated standard cTnT levels for reasons other than MI.

16. The in vitro assay method according to any one of claims 1 to 15, wherein the sample is a blood sample, a serum sample or a plasma sample.

17. A kit for determining the likelihood of MI in a subject, the kit comprising: i) a first binding molecule and a second binding molecule, wherein the first binding molecule is capable of specifically binding to: a cTnT region corresponding to amino acids 201-288 of SEQ ID NO: 1, a cTnI comprising the amino acid sequence shown in SEQ ID NO: 2, a cardiac troponin C (cTnC) comprising the amino acid sequence shown in SEQ ID NO: 3, or an epitope formed when the first group of cTnT molecules is complexed with cTnI and cTnC or when cTnI and cTnC are complexed, and the second binding molecule is capable of specifically binding to a cTnT region corresponding to amino acids 1-200 of SEQ ID NO: 1; or i') a first binding molecule and a second binding molecule, wherein the first binding molecule specifically binds to a cTnT region corresponding to amino acids 195-220 of SEQ ID NO: 1, and the second binding molecule specifically binds to: a cTnT region corresponding to amino acids 1-194 of SEQ ID NO: 1 or a cTnT region corresponding to amino acids 221-288, cTnI, cTnC, an epitope formed when C-terminal cTnT is complexed with cTnI and cTnC, or an epitope formed when cTnI and cTnC are complexed; or ii) a seventh binding molecule capable of specifically binding to: a cTnT region corresponding to amino acids 190-223, preferably amino acids 195-220, of SEQ ID NO: 1, and a detectably labeled cTnT or a peptide having at least a partially intact C-terminal cleavage region thereof.

18. The kit according to claim 17, further comprising a third binding molecule and a fourth binding molecule, wherein the third binding molecule and the fourth binding molecule are capable of specifically binding to different epitopes in the region corresponding to amino acids 69-189 of SEQ ID NO:

1.

19. The kit according to claim 17 or 18, further comprising i) a fifth binding molecule and a sixth binding molecule, the fifth binding molecule and the sixth binding molecule being capable of specifically binding to different epitopes in the amino acid region corresponding to SEQ ID NO: 1, or ii) a fifth binding molecule and a sixth binding molecule, the fifth binding molecule being capable of specifically binding to an epitope in the amino acid region corresponding to SEQ ID NO: 1, and the sixth binding molecule being specifically binding to cTnI, cTnC, or an epitope formed when the cTnT to be measured is complexed with cTnI and cTnC.

20. A kit according to any one of claims 17 to 19, wherein the first binding molecule (if present), the third binding molecule (if present), the fifth binding molecule (if present) and the seventh binding molecule (if present) are capture molecules immobilized on a solid surface.

21. The kit according to any one of claims 17 to 20, wherein the second binding molecule, the fourth binding molecule (if present), the sixth binding molecule (if present) is a tracer molecule comprising a detectable label.

22. A kit according to any one of claims 17 to 20, wherein the first binding molecule (if present), the third binding molecule (if present), the fifth binding molecule (if present) and the seventh binding molecule (if present) are tracer molecules comprising a detectable label.

23. The kit according to any one of claims 17 to 20, wherein the second binding molecule, the fourth binding molecule (if present), the sixth binding molecule (if present) are capture molecules immobilized on a solid surface.

24. Use of a kit according to any one of claims 17 to 23 for determining the likelihood of MI in a subject.

25. Use of a kit according to any one of claims 17 to 23 for distinguishing patients with MI from patients showing elevated standard cTnT levels for reasons other than MI.

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