Methods, reagents, and kits for synchronizing immunoassay signals

Abstract

Methods, assays, systems, and kits for determining the presence, severity, and / or predisposition of heart failure (HF) in an individual are disclosed. The method utilizes a solid substrate in which streptavidin-coated particles are pre-incubated with a biotinylated antibody to form a solid-phase reagent. Since the solid-phase reagent is presented with the biotinylated antibody in a pre-complexed form, the likelihood of biotin from the sample interfering with the assay is low. The methods, systems, assays, and kits of this disclosure enable the NT-proBNP assay to exhibit no biotin interference at biotin concentrations up to 3510 ng / mL.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 514,108, filed on 17 July 2023. All the contents of the patent application referenced above are expressly incorporated herein by reference.

[0002] field This disclosure is in the realm of immunoassays and assay formats suitable for tuning signals by altering their signal-to-noise ratio. Furthermore, this disclosure also relates to stabilizing reagents to eliminate interference from free biotin in biological samples. In certain non-limiting embodiments, this disclosure modifies the signal-to-noise ratio of an NT-proBNP sandwich immunoassay suitable for use in high-throughput analyzers. [Background technology]

[0003] Left ventricular dysfunction can occur as part of coronary heart disease, arterial hypertension, valvular heart disease, and primary cardiomyopathy. If left ventricular dysfunction is left untreated and progressive, it is likely to lead to death, for example, from sudden cardiac death. Chronic heart failure is a clinical syndrome caused by impaired cardiac pumping function. The presence and severity of heart failure are classified into stages I to IV based on symptoms by organizations such as the New York Heart Association (NYHA). Clinical and imaging tests are used to diagnose left ventricular dysfunction.

[0004] Natriuretic peptides reported in the literature include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The importance of natriuretic peptides in regulating cardiovascular function is well established. Biomarkers of brain natriuretic peptide are commonly used to aid in the diagnosis of heart failure (HF) and to assess its severity.

[0005] ANP and BNP have natriuretic and diuretic effects. As antagonists of the renin-angiotensin-aldosterone system, ANP and BNP affect the body's electrolyte and fluid balance. In subjects with left ventricular dysfunction, serum and plasma concentrations of BNP are elevated, as is the concentration of proBNP, a biologically inactive prohormone. proBNP is composed of 108 amino acids. It is primarily secreted by the left ventricle of the heart, where it is cleaved into physiologically active BNP (amino acids 77-108) and the N-terminal fragment NT-proBNP (amino acids 1-76) (see, for example, Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Nadar SK, Shaikh MM., Biomarkers in Routine Heart Failure Clinical Care.Card Fail Rev.2019;5(1):50~56.doi:10.15420 / cfr.2018.27.2 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Biomarkers such as NT-proBNP act as surrogates for clinically meaningful outcomes, sometimes reflecting the underlying etiology of the disease, and sometimes not. Examples of clinical utility include diagnosis, prediction of disease progression or regression, and prediction of mortality. Biomarkers should be easily obtainable, reliable in measurement, and available for continuous monitoring. Ideally, they should also offer the ease of use, timeframe, and / or cost advantages of currently used clinical measures.

[0008] Certain substances in biological samples pose a problem in that they interfere with assay formats for measuring biomarkers. It is well known that biotin present in biological samples can bias assay results and / or interfere with assays in which, for example, a biotinylated antibody is a reagent separate from the solid phase containing biotin-binding molecules, such as (but not limited to) unconjugated streptavidin-coated particles. This assay format has the problem that any naturally occurring biotin in the biological sample has time to form complexes with streptavidin-coated particles, resulting in interference where the biotinylated antibody is blocked from binding to the streptavidin-coated particles.

[0009] Of interest is U.S. Patent No. 8,252,605 by Janzen et al., entitled "Method and Composition For Stabilizing Liquid Reagents" (which is incorporated herein by reference in its entirety). However, this prior art is insufficient in that it does not demonstrate the reduction of biotin interference as shown and described herein, in particular the improvement to the immunoassay for NT-proBNP detection provided herein. [Means for solving the problem]

[0010] Therefore, when the free biotin or natural biotin in question is present or may be present in a biological sample at a concentration sufficient to cause interference, there remains a need in the art for improved assays, such as improved NT-proBNP assays. This disclosure covers such biomarker assay formats, as well as compositions / reagents / kits containing reagents for measuring biomarkers, and methods for using them.

[0011] The embodiments of the present disclosure, briefly summarized above and described in more detail below, can be understood by reference to the exemplary embodiments of the present disclosure shown in the accompanying drawings. However, the accompanying drawings show only typical embodiments of the present disclosure and should not therefore be considered as limiting the scope of the present disclosure, as the present disclosure may admit other equally effective embodiments.

Brief Description of the Drawings

[0012] [Figure 1] FIG. showing a prior art immunoassay format for NT-proBNP. The prior art immunoassay utilizes three reagents: streptavidin-coated particles (Reagent 1), biotinylated antibody (Reagent 2), and acridinium ester-labeled antibody (Reagent 3). The two antibodies can bind to non-overlapping epitopes of NT-proBNP to form a sandwich complex (Product 1). [Figure 2] FIG. showing the effect of biotin interference on the prior art immunoassay of FIG. 1. [Figure 3] FIG. showing a non-limiting embodiment of an NT-proBNP immunoassay constructed in accordance with the present disclosure. In this embodiment, the solid-phase reagent is generated by pre-incubating streptavidin-coated particles with a biotinylated antibody (or a binding fragment thereof) before the addition of the sample. Since the solid-phase reagent is presented with the biotinylated antibody in a pre-formed complex, biotin interference from free biotin or native biotin in the sample is removed or substantially removed from the immunoassay. [Figure 4] FIG. is an exemplary block diagram showing a computer system 1100 suitable for performing the method of the present disclosure with a chemical analyzer. [Figure 5] FIG. is a graph showing a receiver operating characteristic (ROC) curve demonstrating the clinical sensitivity and specificity of an immunoassay constructed in accordance with the present disclosure.

Mode for Carrying Out the Invention

[0013] For ease of understanding, the same reference numbers are used, where possible, to denote the same elements common to the drawings. The drawings are not drawn to scale and may be simplified for clarity. Elements and features of one embodiment may be beneficially incorporated in other embodiments without further elaboration.

[0014] Before detailing at least one embodiment of the present disclosure by way of illustrative representations and results, it is to be understood that the present disclosure is not limited in its application to the details of the construction and arrangement of components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. As such, the expressions used herein are intended to give the broadest scope and meaning possible; embodiments are intended to be illustrative and not exhaustive. Also, it is to be understood that the expressions and terms used herein are for the purpose of description and should not be regarded as limiting.

[0015] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The foregoing methods and procedures are carried out in accordance with conventional methods generally known in the art, as described in various general and more specific references cited and considered throughout this specification. The nomenclature utilized in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry as described herein, as well as their testing procedures and techniques, are well known and commonly used in the art.

[0016] All patents, published patent applications, and non-patent publications referenced herein represent the skill level of a person skilled in the art to which this disclosure relates. All patents, published patent applications, and non-patent publications referenced in any part of this application expressly incorporate them in whole herein to the same extent as each individual patent or publication is specifically and individually indicated to be incorporated by reference.

[0017] All compositions, devices, kits, and / or methods disclosed herein can be prepared and performed without undue experimentation in light of this disclosure. While compositions, devices, kits, and / or methods are described in relation to specific embodiments, it will be apparent to those skilled in the art that modifications can be made to the compositions, devices, kits, and / or methods, as well as to the steps or sets of steps of the methods described herein, without departing from the concepts, spirit, and scope of this disclosure. All such similar substitutes and modifications, apparent to those skilled in the art, shall be deemed to fall within the spirit, scope, and concepts of this disclosure as defined by the appended claims.

[0018] definition When used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated:

[0019] The use of the terms “a” or “an” in the claims and / or specification, when used in conjunction with the term “comprising,” may mean “one,” but is not inconsistent with the meanings of “one or more,” “at least one,” and “one or more.” Therefore, the terms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. Thus, for example, a reference to “one compound” could refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or more compounds. The term “multiple” refers to “two or more.”

[0020] The use of the term "at least one" will be understood to include 1, as well as any two or more quantities, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can be extended to up to 100, 1000, or more, depending on the term it is associated with; in addition, the quantity 100 / 1000 should not be considered limiting, as satisfactory results can be obtained even with a larger upper limit. Furthermore, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0021] The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of distinguishing two or more items and does not, unless explicitly stated otherwise, imply, for example, that one item has any order or significance relative to another, or that there is any additional arbitrary order.

[0022] The use of the term “or” in the claims is used to mean an inclusive “and / or” unless it is explicitly indicated that it refers only to substitutes, or unless the substitutes are mutually exclusive. For example, the condition “A or B” is satisfied by any of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0023] Where used herein, any reference to “one embodiment,” “embodiment,” “some embodiments,” “example,” “for example,” or “example” means that any particular element, feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment. For example, the appearance of the phrase “in some embodiments” or “for example” in various parts of this specification does not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be construed as not limiting the scope of the claims.

[0024] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in errors relating to a composition / apparatus / device, variations in the method used to determine the value, or variations present among the subjects under test. For example, where the term “approximately” is used, the specified value may vary from the specified value by plus or minus 20%, or 15%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, such variations are appropriate for carrying out the disclosed method and will be understood by those skilled in the art.

[0025] The term “antibody” is used herein in its broadest sense and refers to, for example, complete monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and their conjugates exhibiting desired biological activity for binding to an analyte (e.g., Fab, Fab', F(ab')2, Fv, scFv, Fd, diabody, single-chain antibodies, and other antibody fragments and their conjugates that retain at least a portion of the variable region of a complete antibody, etc.), antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. Antibodies may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0026] The terms “biomarker” or “biological marker” are used herein to refer to entities whose presence, level, or form correlates with a particular biological event or condition of interest, and which are consequently considered to be “markers” of that event or condition, in accordance with their usage in the art. To a few examples, in some non-limiting embodiments, a biomarker may be, or include, a marker for a particular medical condition, or for the likelihood that a particular disease, disorder, or condition may develop, occur, or recur. In some non-limiting embodiments, a biomarker may be, or include, a marker for a particular disease or for the outcome or prospect of its treatment. Thus, in some non-limiting embodiments, a biomarker predicts the biological event or condition of interest; in some non-limiting embodiments, a biomarker indicates the prognosis of the biological event or condition of interest; and in some non-limiting embodiments, a biomarker indicates a diagnosis of the biological event or condition of interest. In some non-limiting embodiments, a biomarker is a possible biomarker for the biological event or condition of interest. A biomarker may be an entity of any chemical classification. For example, in some non-limiting embodiments, the biomarker may be a nucleic acid, polypeptide, small molecule, or a combination thereof, or may contain them. In some non-limiting embodiments, the biomarker is a cell surface marker. In some non-limiting embodiments, the biomarker is intracellular. In some non-limiting embodiments, the biomarker is found in a specific tissue (e.g., lung tissue). In some non-limiting embodiments, the biomarker is found extracellularly (e.g., secreted, or otherwise produced or present extracellularly in bodily fluids such as blood, urine, tears, saliva, cerebrospinal fluid, etc.).

[0027] As described herein, in some non-limiting embodiments, the biomarker is an NT-proBNP biomarker. As used herein, “NT-proBNP biomarker” refers to a biological marker for heart failure (HF). In some non-limiting embodiments, one or more NT-proBNP biomarkers include NT-proBNP (amino acids 1-76), which is the N-terminal fragment of proBNP. (See, for example, Non-Patent Document 1).

[0028] The term “characteristic fragment” refers to a fragment of a biomarker (e.g., an NT-proBNP biomarker) that is sufficient to identify the biomarker from which the fragment originated. For example, in some non-limiting embodiments, a “characteristic fragment” of a biomarker includes an amino acid sequence or set of amino acid sequences that collectively enables the distinction of the biomarker from which the fragment originated from other possible biomarkers, proteins, or polypeptides. In some non-limiting embodiments, a characteristic fragment includes at least 10, at least 20, at least 30, at least 40, or at least 50 amino acids. In certain non-limiting embodiments, a characteristic fragment refers to a fragment of a biomarker having at least 90%, at least 95%, or at least 99% sequence identity with respect to the biomarker from which the characteristic fragment originated.

[0029] The term "hybridization" refers to the physical properties of a single-stranded nucleic acid molecule (e.g., DNA or RNA) that anneal to a complementary nucleic acid molecule. Hybridization can be evaluated in a variety of contexts, including when the interacting nucleic acid molecules are isolated or in more complex system contexts (e.g., while covalently or otherwise bound to a carrier entity, and / or within a biological system or cell). In some non-limiting embodiments, hybridization can be detected by hybridization techniques, such as in-situ hybridization (ISH), microarrays, Northern blotting, Southern blotting, and others. In some non-limiting embodiments, hybridization refers to 100% annealing between a single-stranded nucleic acid molecule and a complementary nucleic acid molecule. In some non-limiting embodiments, annealing is less than 100% (e.g., at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, and at least 70% of single-stranded nucleic acid molecules anneal to complementary nucleic acid molecules). Hybridization techniques and methods for evaluating hybridization are well known in the art. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Plainview, NY. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least a desired level of complementarity stably hybridize, while sequences with lower complementarity do not. For examples of hybridization conditions and parameters, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, NJ.

[0030] As used herein, the term "detecting agent" refers to any element, molecule, functional group, compound, fragment or moiety that can be detected. In some non-limiting embodiments, the detecting agent is provided or utilized alone. In some non-limiting embodiments, the detecting agent is provided and / or utilized in conjunction with (e.g., linked to) another agent. Examples of detecting agents include, but are not limited to: various ligands, radionuclides (e.g., 3 H, 14 C, 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes, chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors, nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (e.g., dyes, gold colloids, etc.), biotin, digoxigenin, haptens, and proteins from which antisera or monoclonal antibodies can be obtained.

[0031] As used herein, the term “diagnostic testing” means any process or set of processes performed or carried out to determine whether a patient has a disease, disorder or condition, and / or to obtain information useful in determining the prognosis of a disease, disorder or condition, or in classifying a disease, disorder or condition into a phenotypic category or any category that is significant in terms of the likely response to treatment (either general treatment or any specific treatment). Similarly, the term “diagnosis” means providing any type of diagnostic information, including but not limited to whether a subject has or is likely to develop a disease, disorder or condition; the circumstances, stage, or characteristics of the disease, disorder or condition appearing in the subject; information relating to the nature or classification of a condition, such as tumor or heart disease; information relating to prognosis; and / or information useful in selecting appropriate treatment or additional diagnostic testing. Treatment selection may include selection of a specific therapeutic agent or other form of treatment, such as surgery or radiation therapy; selection of whether to withhold or provide treatment; and selection of a dosing regimen (e.g., the frequency or level of one or more doses of a specific therapeutic agent or combination of therapeutic agents). The selection of additional diagnostic tests may include more specific tests for a given disease, disorder, or condition.

[0032] As used herein and in the claims, the terms “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “includes” and “include”), or “containing” (and any form of “contains” and “contain”) are comprehensive or open-ended and do not exclude additional unlisted elements or process steps. For example, a process, method, article, or apparatus containing a list of elements is not necessarily limited to those elements and may include other elements that are not expressly listed or are not essentially present therein.

[0033] As used herein, the term “or any combination thereof” refers to all permutations and combinations of the items listed preceding the term. For example, “A, B, C, or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and is also intended to include BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, where the order is important in the particular context. Continuing this example, combinations containing one or more repetitions of items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are explicitly included. A person skilled in the art will understand that, unless it is evident from the context otherwise, there is typically no limit to the number of items or terms in any combination.

[0034] As used herein, the term “substantially” means that the event or situation described thereafter occurs entirely, or that the event or situation described thereafter occurs to a considerable extent or degree. For example, in relation to a particular event or situation, the term “substantially” means that the event or situation described thereafter occurs for at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term “substantially adjacent” may mean that two items are 100% adjacent to each other, or that two items are close to each other but not 100% adjacent, or that a portion of one of the two items is close to the other but not 100% adjacent to the other.

[0035] As used herein, the phrase “bonded to” includes both direct and indirect bonding of two parts to each other. Non-limiting examples of bonding include covalent bonding of one part to another, either by direct bonding or via spacer groups; non-covalent bonding of one part to another, either directly or by members of specific bond pairs bonded to the part; incorporation of one part into another, such as by dissolving or synthesizing one part into another; and coating one part onto another.

[0036] As used herein, the term “biological fluid sample” will be understood to include any liquid test sample obtained from a patient and available in accordance with this disclosure. Examples of available biological fluid samples include, but are not limited to, whole blood or any part thereof (i.e., plasma or serum), serum, EDTA plasma, lithium heparin plasma, and combinations thereof.

[0037] As used herein, the term “volume” in the context of liquid test samples used in accordance with this disclosure typically refers to the volume of a liquid test sample, such as a range of about 0.1 μl to about 100 μl, or a range of about 1 μl to about 75 μl, or a range of about 2 μl to about 60 μl, or a value of about 50 μl or less.

[0038] As used herein, the term “patient” includes humans and veterinary subjects. In certain non-limiting embodiments, the patient is a mammal. In certain other non-limiting embodiments, the patient is a human. For diagnostic / procedural purposes, the term “mammal” refers to any animal classified as a mammal, including humans, domesticated and livestock, non-human primates, and zoo animals, sports animals, or companion animals, such as dogs, horses, cats, and cows.

[0039] "Healthcare provider" or "healthcare decision-maker" includes any individual authorized to diagnose or treat a patient, or to assist in the diagnosis or treatment of a patient. In the context of identifying a useful new drug to treat a particular disease, disorder, or condition, the healthcare provider may be an individual not authorized to diagnose or treat a patient, or to assist in the diagnosis or treatment of a patient.

[0040] A "point-of-care test" refers to a real-time diagnostic test that can be performed within a rapid timeframe, so that the resulting test is performed faster than an equivalent test that does not use this system. Point-of-care tests can be performed quickly and on-site, especially where rapid and accurate results are required, such as in a clinic, bedside, emergency room, emergency treatment room, or other such locations. Patient presence is possible but not required. Point-of-care includes, but is not limited to, emergency rooms, operating rooms, hospital laboratories and other clinical laboratories, clinics, on-site, or any situation where rapid and accurate results are desired.

[0041] The term “specific binding partner” will be understood to refer to any molecule that can specifically bind to a target analyte, especially (but not limited to) when used herein in the terms “target analyte specific binding partner” or “biotin specific binding partner.” For example, but not limited to, a binding partner may be an antibody, receptor, ligand, aptamer, molecular imprinted polymer (i.e., inorganic matrix), a combination or derivative thereof, as well as any other molecule that can specifically bind to a target analyte.

[0042] As used herein, the term “immunoassay” refers to an assay for determining the presence of a diagnostic biomarker in a biological sample by reacting the sample with an antibody (or fragment thereof) that specifically binds to the diagnostic biomarker or a characteristic fragment thereof, wherein the reaction is carried out under conditions and for a time that allows for the formation of an immune complex between the antibody (or fragment thereof) and the diagnostic biomarker. Subsequently, such an immune complex is quantitatively measured.

[0043] Sample: As used herein, the term “sample” means a biological sample obtained from or derived from a human subject, as described herein. In some embodiments, a biological sample includes biological tissue or biological fluid. In some embodiments, a biological sample may include blood; blood cells; tissue or fine-needle biopsy specimens; cell-containing fluids; suspended nucleic acids; cerebrospinal fluid; lymph; tissue biopsy specimens; surgical specimens; other body fluids, secretions and / or excretions; and / or cells derived therefrom. In some embodiments, a biological sample includes cells obtained from an individual, for example, from a human or animal subject. In some embodiments, the cells obtained are cells from the individual from which the sample is obtained, or include those cells. In some embodiments, a sample is a “primary sample” obtained directly from the source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, or collection of body fluid (e.g., blood). In some embodiments, a sample is cardiac tissue obtained from a subject. In some embodiments, as will be apparent from the context, the term “sample” refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). For example, this may be by filtration using a semipermeable membrane. Another example of sample processing is that the sample may be a plasma sample treated with an anticoagulant selected from the group consisting of EDTA, heparin, and citrate. Another example of sample processing is that the sample is processed to isolate one or more proteins (e.g., by capturing proteins with one or more antibodies). A “processed sample” may include, for example, nucleic acids or polypeptides extracted from the sample or obtained by subjecting the primary sample to a method such as mRNA amplification or reverse transcription, isolation and / or purification of certain components.

[0044] Subject: As used herein, the term “subject” means an organism, e.g., a mammal (e.g., human). In some embodiments, a human subject is an adult, adolescent, or child subject. In some embodiments, a subject is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. In some embodiments, a subject suffers from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to developing a disease, disorder, or condition and / or exhibits an increased risk thereof (compared to the mean risk observed in a reference subject or population). In some embodiments, a subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any particular symptoms or features of a disease, disorder, or condition (e.g., clinical symptoms of a disease). In some embodiments, a subject does not exhibit any symptoms or features of a disease, disorder, or condition, such as heart failure. In some embodiments, a subject is a patient. In some embodiments, the subject is an individual to be diagnosed and / or treated.

[0045] Threshold: As used herein, the term “threshold” refers to a value (or set of values) used as a reference to obtain information about the results of a measurement, for example, the results of a measurement achieved in an assay, and / or to classify them. A threshold may be determined based on one or more control samples. A threshold may be determined before, at the same time as, or after the measurement of interest is performed. In some embodiments, a threshold may be a range of values. In some embodiments, a threshold may be a value (or range of values) reported in the relevant field (e.g., a value found in a standard table).

[0046] The term "stratification" refers to dividing a potential patient population, or a patient population, into subgroups such as strata or blocks. In various embodiments, a "strata" refers to a specific division of the patient population.

[0047] Turning to various non-limiting embodiments of this disclosure, improved biomarker assay formats, as well as compositions / reagents / kits containing reagents for measuring biomarkers, along with methods for using them, are described herein. In certain non-limiting embodiments, this disclosure provides improved assays, such as (but not limited to), improved NT-proBNP assays, which have improved performance when the free biotin or natural biotin in question is present or may be present in a biological sample and may cause interference with existing assay formats. In some embodiments, the biomarker assay format is designed to adjust the signal from the signal portion.

[0048] In certain non-limiting embodiments, the assay format includes one or more streptavidin-coated solid supports (but not limited to one or more bead substrates or particles) pre-incubated with one or more biotinylated antibodies (or their binding fragments) to form one or more solid-phase reagents suitable for use in an immunoassay, while eliminating or substantially eliminating interference from free biotin in the biological sample. In certain non-limiting embodiments, the disclosure modifies the signal-to-noise ratio of an NT-proBNP sandwich immunoassay suitable for use in a high-throughput analyzer. In certain non-limiting embodiments, there is substantially no opportunity for free biotin or natural biotin from the sample to interfere with the assay because the solid-phase reagent is presented with the biotinylated antibody in a pre-complexed form. In certain non-limiting embodiments, such as NT-proBNP analytes, “no interference to biotin” is defined as a percentage bias of 10% or less. In certain non-limiting embodiments, the present disclosure allows us to claim that the NT-proBNP assay is uninterference-free up to biotin levels of 3510 ng / mL.

[0049] In certain non-limiting embodiments, the immunoassay can be performed on a high-throughput chemical analyzer capable of performing more than 20, 30, 40, 50, 75, 100, or more tests per hour. In certain non-limiting embodiments, the disclosure also includes a non-transient computer-readable medium suitable for performing the method of the disclosure on a chemical analyzer.

[0050] In certain (but non-limiting) embodiments, the immunoassay can detect a complex between a serum marker or a characteristic fragment thereof and a serum marker-conjugating antibody using a second antibody that is labeled and also conjugates to the serum marker or fragment thereof. In certain certain non-limiting embodiments, a sandwich immunoassay is used in which the serum marker-conjugating antibody may be a capture antibody bound to an insoluble material (but not limited to magnetic beads), and the second antibody may be a labeled antibody. The above sandwich immunoassay procedure can be used with the antibodies described below herein.

[0051] Turning now to certain non-limiting embodiments of the present disclosure, embodiments of the present disclosure include assay formats suitable for detecting individual biomarkers such as NT-proBNP or characteristic fragments thereof, as well as compositions / devices / kits containing the same, methods for manufacturing and using the same, kits, and diagnostic tests associated therewith. Certain non-limiting embodiments of the present disclosure include pre-selected assay formats for detecting NT-proBNP in serum. Certain non-limiting embodiments of the present disclosure favorably reduce, eliminate, or substantially eliminate biotin interference in the reaction. In certain non-limiting embodiments, the pre-selected assay format favorably limits the binding of biotin to the reagents of the assay.

[0052] Certain non-limiting embodiments of this disclosure relate to a method for determining the presence, severity, and / or predisposition of heart failure in an individual using a single biomarker. In certain non-limiting embodiments, this disclosure relates to a method having a set of steps, including (a) forming a solid-phase immunoassay reagent by contacting one or more streptavidin-coated particles with one or more biotinylated antibodies to form a complex; and (b) separating the solid-phase immunoassay reagent from the immunoassay Wright reagent and storing or packaging the solid-phase reagent separately from the Wright reagent. In certain non-limiting embodiments, the solid-phase reagent is presented with the biotinylated antibody in a pre-complexed form so that there is virtually no opportunity for biotin from the biological fluid sample to interfere with the assay. With respect to the NT-proBNP analyte, “no biotin interference” is defined as a percentage bias of 10% or less. In certain non-limiting embodiments of this disclosure, the term “no biotin interference” includes no interference at biotin concentrations up to 3510 ng / mL.

[0053] This disclosure facilitates the diagnosis of HF, such as point-of-care or telediagnosis, and helps healthcare providers monitor the status or progression of HF at two or more points in time. Embodiments of this disclosure are suitable for use in an outpatient setting, for example, when a patient who requires it provides a biological sample, or when one or more embodiments of this disclosure are used as part of an outpatient care plan, for example, when embodiments of the medical service of this disclosure do not require hospitalization, or when the patient may freely leave the medical facility once the service or procedure of this disclosure is completed. Non-limiting examples of outpatient care include home healthcare services, physician's clinics, or annual health checkups in non-hospital setting (e.g., clinics, outpatient surgery centers, hospital outpatient departments, and other non-hospital facilities). In some embodiments, embodiments of this disclosure are suitable for use by users in home outpatient care to promote patient independence, enable a transition from hospital to home care, or as part of a home monitoring plan, such as cardiac monitoring. In some embodiments, the assays of this disclosure can be applied in a single step in an outpatient care setting, such as an outpatient facility, where the patient does not need to be present for more than 1 to 24 hours, more than 24 hours, or more than 12 hours.

[0054] In some embodiments, a patient requiring such a biological sample, such as blood or serum, is provided from an outpatient setting. The biological sample can be shared with a facility suitable for performing one or more assays or embodiments of the present disclosure in order to analyze the patient's health information, such as cardiac information. In some embodiments, after sample collection, a chemical reaction between the analytes and reagents in the patient's biological sample is carried out by contact under conditions suitable for performing the assay of the present disclosure, resulting in the generation of various or adjusted signals that are measured by an analyzer.

[0055] Kits comprising one or more anti-HF biomarker agents and instructions for use (e.g., treatment, prophylactic, or diagnostic use) are also provided by this disclosure. In some non-limiting embodiments, the kit is used for an in vitro diagnostic assay for diagnosing HF. In some non-limiting embodiments, one or more anti-HF biomarker agents comprise antibody agents. In some non-limiting embodiments, one or more of the antibody agents are labeled with a detectable portion. In some non-limiting embodiments, the kit further comprises a detection agent (e.g., one or more acridinium ester molecules). In some non-limiting embodiments, one or more of the antibody agents are labeled with one or more acridinium ester molecules. In some non-limiting embodiments, the kit further comprises one or more secondary antibody agents that specifically bind to one or more anti-HF biomarker antibody agents. Further embodiments of the kit are described in the following examples.

[0056] In certain non-limiting embodiments, the instructions for use provide suitable usage methods for the ATELLICA® CI Analyzer (Siemens Healthineers USA, Malvern, PA), for example, the 1900 model.

[0057] In some non-limiting embodiments, the kit further includes one or more control samples. In some non-limiting embodiments, the control samples include one or more HF biomarker reference materials.

[0058] In addition to the above, the kit may include other components such as (but not limited to) solvents or buffers, stabilizers or preservatives, and / or agents for treating the conditions or disorders described herein. Alternatively, the other components may be included in the kit in a separate composition or container from the anti-HF biomarker agent. In such embodiments, the kit may include instructions for mixing the anti-HF biomarker agent with the other components, or for using the anti-HF biomarker together with the other components. In certain non-limiting embodiments, the provided instructions eliminate the time for any biotin in the sample to form a complex with streptavidin-coated particles and / or prevent the biotin-labeled antibody from binding to the streptavidin-coated particles. In certain non-limiting embodiments, the instructions include using streptavidin-coated particles that are pre-incubated with a biotinylated antibody and characterized as being in the form of a solid-phase reagent.

[0059] In certain non-limiting embodiments, a kit for use in accordance with this disclosure may include a reference or control sample, instructions for processing the sample, instructions for performing tests on the sample, and / or instructions for interpreting the results, as well as buffers and / or other reagents necessary for performing the tests.

[0060] The methods and kits provided herein detect NT-proBNP in a sample with sensitivity and specificity sufficient to make the test results medically usable. The methods and kits described herein for the detection and / or diagnosis of HF in a subject detect NT-proBNP with sensitivity greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or about 100%. In some non-limiting embodiments, the methods and kits provided herein can detect NT-proBNP with sensitivity between about 70% and 100%, between about 80% and 100%, or between about 90% and 100%. In some non-limiting embodiments, the methods and kits provided herein can detect NT-proBNP with sensitivity and specificity between approximately 50% and 100%, between approximately 60% and 100%, between approximately 70% and 100%, between approximately 80% and 100%, or between approximately 90% and 100%.

[0061] Compositions are also provided herein. In some non-limiting embodiments, the composition includes one or more biotinylated antibodies bound to one or more streptavidin-coated solid supports (e.g., streptavidin-coated beads or particles, but not limited thereto). In certain non-limiting embodiments, the assay format includes one or more streptavidin-coated particles, e.g., one or more bead substrates pre-incubated with one or more biotinylated antibodies, for forming one or more solid-phase reagents suitable for use in removing or substantially removing interference from free biotin in a biological fluid sample. In certain non-limiting embodiments, the disclosure modifies the signal-to-noise ratio of an NT-proBNP sandwich immunoassay suitable for use in high-throughput analyzers. In certain non-limiting embodiments, the solid-phase reagent is presented with the biotinylated antibody in a pre-complexed form so that when the reagent is mixed with the sample, there is virtually no opportunity for free biotin from the sample or native biotin to interfere with the assay. In certain non-limiting embodiments, such as those for NT-proBNP analytes, “no interference to biotin” is defined as a percentage bias of 10% or less. In certain non-limiting embodiments, the present disclosure allows the NT-proBNP assay to claim no interference up to biotin levels of up to 3510 ng / mL.

[0062] In certain non-limiting embodiments, the immunoassay can be performed on a high-throughput chemical analyzer capable of performing more than 20, 30, 40, 50, 75, or 100 tests per hour. In certain non-limiting embodiments, the immunoassay can be performed on, for example, an ATELLICA® CI Analyzer (Siemens Healthineers USA, Malvern, PA), such as the 1900 model.

[0063] In immunoassays, a form of diagnostic method, one or more specific conjugates are used. A typical example is a sandwich immunoassay, in which two specific conjugates (antibodies or antigens) bind to non-overlapping epitopes of the analyte of interest. One of the specific conjugates is usually linked to a so-called label or tag, which can be an atom (e.g., radioactive), a molecule (e.g., an enzyme, fluorescent or luminescent compound), or a particle (magnetic or latex). This label enables the detection of the analyte of interest through various detection methods corresponding to the label used.

[0064] Other specific binding species are often covalently or adsorbently bound to a solid or suspended substrate ("solid phase"). Alternatively, they may be linked to the first member of a second binding pair (e.g., biotin), while the second member of the second binding pair (e.g., streptavidin) is bound to the solid phase. This allows the specific binding species to bind to the solid phase via the interaction of the second binding pair (e.g., biotin-streptavidin).

[0065] The solid phase may be a solid phase visible to the naked eye, such as a microtiter well, tubes and balls in a tubing apparatus (but not limited to these), or a suspended solid phase, such as beads, latex beads, magnetic latex beads, etc. (but not limited to these), as well as other paramagnetic materials. Secondary bond species are generally labeled using tags. The interaction between the tag and the solid phase enables the detection and quantification of the analyte of interest via various detection methods corresponding to the labels used.

[0066] Figures 1 and 2 show prior art sandwich immunoassays for analytes such as NT-proBNP (but not limited to it), along with the effect of biotin interference. As seen in Figure 1, the prior art assay utilizes three reagents: Reagent 1 is streptavidin-coated particles (e.g., streptavidin-coated magnetic beads, but not limited to them); Reagent 2 is a biotinylated first antibody; and Reagent 3 is an acridinium-labeled second antibody. The two antibodies, Reagents 2 and 3, bind to non-overlapping epitopes of NT-proBNP, so that both antibodies can bind to a single molecule of NP-proBNP. When these three reagents are mixed with a biological sample suspected of containing NT-proBNP (e.g., plasma or serum, but not limited to these), the two antibodies bind to NP-proBNP, and the biotinylated antibody binds to streptavidin in the particles to form reaction product 1, in which the acridinium ester indirectly binds to the solid phase particles via the binding of the two antibodies to NT-proBNP. Therefore, reaction product 1 can be detected via the signal generated by the acridinium ester bound to the solid phase.

[0067] However, as shown in Figure 2, if free biotin is present in the biological sample, the free biotin competes with the biotinylated antibody for binding to the streptavidin-coated particles. As a result, three reaction products are produced: reaction product 1, which is detectable; reaction product 2, which contains NP-proBNP molecules bound by both antibodies (and therefore contains acridinium ester); and reaction product 3, which contains free biotin bound to the streptavidin-coated particles. Consequently, the acridinium ester labels bound to the two antibodies and the NT-proBNP analyte (product 2) cannot bind to the streptavidin-coated particles (product 3) for label detection. Therefore, some of the NT-proBNP present in the sample is not present in product 1 and is therefore not detected, leading to an incorrectly low (or falsely negative) measurement.

[0068] In contrast, Figure 3 shows non-limiting embodiments of kits, systems, and methods for performing immunoassays of target analytes (e.g., NT-proBNP, but not limited to those) according to the present disclosure. In this embodiment, only two reagents are used: Reagent 1 is streptavidin-coated particles pre-bound with a biotinylated first antibody, and Reagent 2 is an acridinium-labeled second antibody. The use of these two reagents ensures that free biotin present in the sample does not interfere, or substantially interferes, with the formation of product 1, in which the binding of NT-proBNP by the two antibodies links the acridinium ester label to the solid-phase particles.

[0069] In certain (but non-limiting) embodiments, the use of streptavidin-coated magnetic beads results in a fast magnetic response time and is suitable for use in high-throughput and multiplex assays. The streptavidin coating is contacted with one or more biotinylated antibodies under conditions suitable for binding the streptavidin coating to the biotinylated antibody. Thus, the immunoassay of this disclosure may be a fully automated two-site sandwich immunoassay using direct chemiluminescence technology, using a fixed or substantially fixed amount of two monoclonal antibodies. The solid phase contains a conjugated biotinylated monoclonal sheep anti-human antibody that is specific to NT-proBNP and conjugated with streptavidin magnetic particles. The Wright reagent contains an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment that is specific to NT-proBNP. In certain non-limiting embodiments, there is a direct relationship between the amount of NT-proBNP present in a patient sample and the amount of relative luminescence units (RLU) detected by the system.

[0070] The methods described herein can be implemented in a computer system having a processor that executes specific instructions in a computer program. In some non-limiting embodiments, the computer system can be configured to output an HF biomarker score based on the reception of an HF biomarker profile and / or levels of an NT-proBNP biomarker. In particular, the computer program may include instructions for the system to select an appropriate next step, including additional drug therapy, treatment, and / or additional tests for the subject.

[0071] In some non-limiting embodiments, a computer system can be configured with a computer program that, based on received data (e.g., NT-proBNP biomarker profiles), identifies subjects for further examination (e.g., cardiac examination), identifies subjects at risk of having HF or who have HF, and / or subjects who should receive drug therapy, and uses that data to calculate an NT-proBNP biomarker score.

[0072] Figure 4 is a block diagram of a computer system 1100 that can be used in the operation described above, according to one non-limiting embodiment. The system 1100 includes a processor 1110, memory 1120, storage device 1130, and input / output device 1140. Each of the components 1110, 1120, 1130, and 1140 is interconnected using a system bus 1150. The system may optionally further include an analytical instrument 1160 for determining the level of one or more biomarkers of this disclosure in a sample.

[0073] In certain non-limiting embodiments, the processor 1110 can process instructions for execution within the system 1100. In one non-limiting embodiment, the processor 1110 is a single-threaded processor. In another non-limiting embodiment, the processor 1110 is a multi-threaded processor. The processor 1110 can process instructions stored in memory 1120 or storage device 1130, including receiving or transmitting information via input / output device 1140.

[0074] In certain non-limiting embodiments, memory 1120 stores information within system 1100. In one non-limiting embodiment, memory 1120 is a computer-readable medium. In one non-limiting embodiment, memory 1120 is a volatile memory unit. In another embodiment, memory 1120 is a non-volatile memory unit.

[0075] The storage device 1130 can provide large-capacity storage to the system 1100. In one non-limiting embodiment, the storage device 1130 is a computer-readable medium.

[0076] The input / output device 1140 provides input / output operation for the system 1100. In one non-limiting embodiment, the input / output device 1140 includes a keyboard and / or a pointing device. In one non-limiting embodiment, the input / output device 1140 includes a display unit for displaying a graphical user interface.

[0077] System 1100 can be used to build a database. In certain non-limiting embodiments, the method of this disclosure is performed in System 1100 located within a chemical analyzer. For example, a computer program product may include instructions causing the processor 1110 to perform any of the steps disclosed herein or otherwise assumed.

[0078] Furthermore, a non-temporary computer-readable medium is provided which, when executed, causes a processor to perform an operation including the methods provided herein. For example, the non-temporary computer-readable medium includes, when executed, an executable instruction that causes a processor to perform an operation including any of the methods disclosed herein or otherwise assumed. In some non-limiting embodiments, the non-temporary computer-readable medium includes hard drives, external hard disks, disks, CDs, DVDs, etc., for storing data. In certain non-limiting embodiments, software located on a physical medium is preferred for use herein.

[0079] In some non-limiting embodiments, a non-transient computer-readable medium is disclosed which includes executable instructions causing a processor to perform an action that, if executed, includes a method for determining the presence, severity, and / or predisposition of heart failure (HF) in an individual, the method including the steps of incubating the assay components of the Disclosure with a biological sample and determining the amount of NT-proBNP in the sample. [Examples]

[0080] Examples are presented below. However, it should be understood that this disclosure is not limited in its application to the specific experiments, results, and test procedures disclosed below. The examples are provided not as an exhaustive but merely as one of various embodiments. [Examples]

[0081] This disclosure includes, in non-limiting embodiments, NT-proBNP assays suitable for in vitro diagnostic applications in the quantitative determination of N-terminal pro-brain natriuretic peptide (NT-proBNP) in human serum and plasma (EDTA and lithium heparin) using chemical analyzers such as (but not limited to) the ADVIA Centaur® XP system. In emergency department (ED) and outpatient (OP) populations, NT-proBNP measurements are used to aid in the diagnosis of heart failure (HF) in patients with clinical suspicion of new-onset or exacerbation of HF, and for assessing the severity of HF.

[0082] In one non-limiting embodiment, the materials present in the kit include: • Wright Reagent (7.5 ml / reagent pack) containing acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment antibody (approximately 0.36 μg / ml); bovine serum albumin (BSA); bovine gamma globulin; and preservatives in a buffer. A solid-phase reagent (20.0 ml / reagent pack) containing a buffer solution containing biotin-labeled monoclonal sheep anti-human NT-proBNP antibody (approximately 2 μg / ml), BSA, bovine gamma globulin, sheep gamma globulin, and a preservative, bound to streptavidin magnetic particles (approximately 220 mg / L). • Any auxiliary well reagent (7.5 ml / reagent pack) containing buffer solution, BSA, bovine gamma globulin, sheep gamma globulin, and preservatives.

[0083] In certain non-limiting embodiments, the assay of the present disclosure is a fully automated two-site sandwich immunoassay using direct chemiluminescence technology, employing fixed amounts of two monoclonal antibodies. The solid phase is specific to NT-proBNP and contains conjugated biotinylated monoclonal sheep anti-human antibodies with streptavidin magnetic particles. The Wright reagent contains an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP. A direct relationship exists between the amount of NT-proBNP present in the patient sample and the amount of relative light units (RLU) detected by the system.

[0084] Non-limiting procedures for collecting specimens or samples may include one or more of the following steps: observe standard precautions when collecting specimens; treat all specimens as potentially disease-transmitting; follow recommended procedures for collecting diagnostic blood specimens by venipuncture; follow the instructions provided with the specimen collection device for use and handling; ensure blood specimens are completely coagulated before centrifugation; and / or keep the tubes capped at all times.

[0085] Non-limiting procedures for storing specimens or samples may include one or more of the following steps, depending on the circumstances: Serum specimens stored on blood clots after centrifugation are stable for up to 24 hours at 2–8°C; Separated samples are stable for up to 3 days at room temperature and up to 4 days at 2–8°C; Separated samples are stable for up to 12 months below -20°C; Avoid more than two freeze-thaw cycles; Do not store in a defrost-free freezer; and / or use after thoroughly mixing thawed samples and centrifugating them.

[0086] Non-limiting procedures for transporting specimens or samples may include, depending on the circumstances, one or more of the following steps: packing and labeling the specimen for transport in accordance with applicable federal and international regulations relating to the transport of clinical specimens and pathogens; and / or transporting the specimen frozen if it may be exposed to temperatures above 25°C during transport.

[0087] The non-limiting procedure for preparing the sample may include one or more of the following steps: This assay requires 20 μL of sample per measurement. This volume does not include any unusable volume in the sample container or any additional volume required for repeated or other tests on the same sample; the sample volume required for onboard dilution is different from the sample volume required for a single measurement on an undiluted sample; do not use obviously contaminated samples; before placing the sample in the system, ensure that the sample is free of: air bubbles or foam, fibrin or other particulate matter; and / or remove particulate matter by centrifugation as recommended by the CLSI guidance and the manufacturer of the sampling device.

[0088] A non-limiting assay procedure may include one or more of the following steps: The system (including a high-throughput chemistry analyzer) automatically performs the following steps: dispense 20 μL of sample into a cuvette; dispense 200 μL of solid phase and 75 μL of auxiliary well reagent, followed by incubation at 37°C for 3 minutes; dispense 75 μL of Wright reagent, followed by incubation at 37°C for 6 minutes; perform a series of washes using a washing reagent (e.g., ADVIA Centaur Wash 1, but not limited to this); dispense 300 μL each of acid and base reagents (e.g., ADVIA Centaur Acid Reagent and ADVIA Centaur Base Reagent, but not limited to these) to initiate the chemiluminescence reaction; and / or report the results.

[0089] Non-limiting instructions for preparing reagents for use in the automated system include: all reagents are liquid and ready for use; reagents must be mixed before loading the packs into the system; refer to the system's online help for information on reagent mixing.

[0090] Non-limiting instructions for preparing the automated system to perform the assays described herein include: ensuring that sufficient materials are loaded into the system; and referring to the materials provided and any materials that may need to be provided separately for guidance on the required reagents.

[0091] Non-restrictive instructions for setting the master curve definition include: entering assay master curve values ​​by scanning the master curve card before starting calibration for each new lot of reagent.

[0092] Non-limiting instructions for calibration include: using the calibration reference material provided with each kit for calibration of a specific, non-limiting embodiment of the assay; the calibration reference material provided in the assay kit must be used only with the reagent lot provided in the same kit.

[0093] Non-limiting instructions regarding calibration frequency include: performing calibration when one or more of the following conditions exist: at the end of a 36-day calibration interval; when changing the lot number of the primary reagent pack; when instructed by quality control results; and after major maintenance or service, when instructed by quality control results. Comply with government regulations or accreditation requirements regarding calibration frequency. Individual laboratory quality control programs and procedures may require more frequent calibration.

[0094] Non-limiting instructions for preparing calibration standards include one or more of the following steps, depending on the circumstances: Add 2.0 mL of reagent water to each vial. Replace the cap. Allow the vial to stand at room temperature for 30 minutes to dissolve the lyophilized material. Gently mix the vials and invert them to ensure homogeneity of the material. Divide into smaller portions and seal them for long-term storage. Store the reconstituted material according to the stability limits specified in the Reagents section. Do not store in a freezer that does not require frosting. Thaw the material completely before using frozen calibration standards. Gently mix the vials and invert them to ensure homogeneity of the material. Use the calibration standards within the stability limits specified in the Reagents section and discard all remaining material.

[0095] Non-limiting instructions for performing the calibration procedure include: ensuring that the appropriate master curve and values ​​assigned to the calibration standards are entered into the system. Refer to the system's online help for information on the definition of the master curve and the input of calibration standard values. Load the reagents required for the assay. Schedule the calibration standards. Label two sample containers with barcode labels: one for the low concentration of calibration standards and the other for the high concentration. Place the barcode labels on the sample containers so that the legible characters are vertical. Barcode labels are lot-specific. Barcode labels from one lot of calibration standards should not be used with any other lot of calibration standards. Gently mix the products and dispense a sufficient volume of each calibration standard into the appropriate sample container. Avoid air bubbles. The sample volume required for the test depends on several factors. Refer to the system's online help for information on sample volume requirements. Load the samples according to the system's online help. Discard any calibration standards remaining in the sample containers after 6 hours. Sample containers must not be refilled or reused. Calibration standard materials must not be returned to their original containers.

[0096] Non-limiting instructions for performing quality control include: using appropriate quality control materials at least once during each day of sample analysis for quality control of a particular non-limiting embodiment of the assay of this disclosure, with known analyte concentrations having at least two levels (low and high); using quality control materials in accordance with the quality control instructions; using additional quality control materials at the discretion of the laboratory; using quality control materials in accordance with the quality control instructions; and in addition, performing quality control in the following cases: following valid calibration; using a new lot of reagent; and troubleshooting test results that do not correspond to a clinical condition or symptom; complying with government regulations or accreditation requirements regarding the frequency of quality control; individual laboratory quality control programs and procedures may require more frequent quality control testing; acceptable performance is achieved when the obtained analyte values ​​are within the expected control interval of the system, as indicated by the manufacturer of the control material, or within the interval determined by the laboratory's quality control procedures; and if the obtained results are not within acceptable limits, follow the laboratory's quality control procedures. For information regarding the input of quality control definitions, see the system's online help.

[0097] Non-limiting instructions for taking corrective actions include: not reporting results if the quality control results fall outside the expected control range; and taking corrective actions according to established laboratory protocols. Refer to the system's online help for suggested protocols.

[0098] result Calculation of the result The system determines the results using the calculation procedures described in the system's online help. The system reports results in pg / mL (common unit) or pmol / L (SI unit), depending on the units defined at the time the assay is set up. Conversion formula: 1 pg / mL = 0.118 pmol / L. For information regarding results outside of the specified measurement interval, please refer to the Measurement Interval section.

[0099] Dilution The measurement interval is 35–35,000 pg / mL (4.13–4130 pmol / L). For information on dilution options, please refer to the system's online help. To obtain accurate results, dilute and retest samples with NT-proBNP levels above 35,000 pg / mL (4130 pmol / L). For automated dilution, perform the following: Load a multi-purpose diluent (e.g., ADVIA Centaur Multi-Diluent 1, but not limited to this). Ensure that sufficient sample volume is available. Refer to the table below. Select an appropriate dilution factor. For automated dilution, enter a dilution point below 35,000 pg / mL (4130 pmol / L).

[0100] [Table 1]

[0101] If the patient's results exceed the assay interval when using automated dilution, or if the laboratory protocol requires manual dilution, the patient sample should be diluted manually. For manual dilution, perform the following: Manually prepare the diluent using a multi-purpose diluent (e.g., ADVIA Centaur Multi-Diluent 1, but not limited to vials). In various embodiments, refer to the operating manuals and considerations relevant to any material. For information regarding ordering tests for manually diluted samples, refer to the system's online help. Ensure that the results are mathematically corrected for dilution. The system automatically calculates the results when the dilution factor is entered during test scheduling.

[0102] Interpretation of results The results from the assays described herein should be interpreted in conjunction with the patient's medical history, clinical findings, and other findings, using appropriate clinical guidelines. These guidelines recommend the use of natriuretic peptides in both emergency department (ED) and outpatient (OP) settings for the diagnosis or exclusion of heart failure. The performance of the assays described herein was evaluated separately in each of these settings using the published age-independent and age-dependent cutoff values.

[0103] Emergency Department (ED) Group For patients who presented to an ED environment with clinically suspected HF, the results of the NT-proBNP assay should be interpreted as shown in Table 2.

[0104] [Table 2]

[0105] Elevated or decreased natriuretic peptide levels can be caused by conditions that may complicate the diagnosis of heart failure. In ED, conditions such as chronic heart failure, acute coronary syndrome, atrial fibrillation, pulmonary embolism, valvular heart disease, myocarditis, pulmonary hypertension, renal failure, stroke, and sepsis can increase NT-proBNP levels in the absence of acute heart failure. Furthermore, obesity, acute pulmonary edema, pericarditis, and cardiac tamponade are associated with decreased NT-proBNP levels.

[0106] Outpatient population In an outpatient setting, optimal use of the natriuretic peptide assay can rule out HF. Therefore, a lower rule-out cutoff value is needed to increase sensitivity and negative predictive value, as these patients may present with limited and less acute HF symptoms. For outpatients who visit an outpatient facility with clinical suspicion of previously undiagnosed HF, the assay results should be interpreted as shown in Table 3.

[0107] [Table 3]

[0108] Clinical conditions such as acute coronary syndrome, atrial fibrillation, pulmonary embolism, valvular heart disease, myocarditis, pulmonary hypertension, renal failure, stroke, and sepsis may increase NT-proBNP levels in the absence of heart failure.

[0109] Restrictions Patient samples may contain heterophile antibodies that react with immunoassays, potentially causing falsely elevated or decreased results. This assay is designed to minimize interference from heterophile antibodies. Further information may be required for diagnosis.

[0110] Expected values ​​in healthy populations (Table 4) Expected values ​​were nonparametrically established for a population of 723 apparent healthy subjects (362 women and 361 men) without HF, using the ADVIA Centaur® XP system in accordance with CLSI document EP28-A3c.

[0111] [Table 4]

[0112] As with all in vitro diagnostic assays, each laboratory should determine its own reference ranges for the diagnostic evaluation of patient results. These values ​​should be considered for guidance only.

[0113] Disease research group ED population: A total of 3128 subjects who presented with signs and symptoms of HF and sought treatment for ED were enrolled. Of these, 1148 subjects (476 women and 672 men) were diagnosed with acute HF and included in the disease study group. NT-proBNP values ​​for subjects with acute HF are summarized in Tables 5-6 by age group (Table 5) and subgroups based on NYHA functional classification (Table 6). Table 7 summarizes the data for men with acute HF, and Table 8 summarizes the data for women with acute HF.

[0114] [Table 5]

[0115] [Table 6]

[0116] [Table 7]

[0117] [Table 8]

[0118] The ED population includes all subjects with acute HF (e.g., NYHA functional classification). The NYHA classification of the ED population with acute HF is shown in Table 9. Men with acute HF (NYHA functional classification) are shown in Table 10, and women with acute HF are shown in Tables 11-12.

[0119] [Table 9]

[0120] [Table 10]

[0121] [Table 11]

[0122] [Table 12]

[0123] Outpatient (OP) group A total of 1033 subjects who presented with signs and symptoms of HF in an OP setting were enrolled. Of these, 185 subjects (102 women and 83 men) were diagnosed with new-onset HF and included in the disease study group. NT-proBNP values ​​for subjects with new-onset HF are summarized based on age group (Tables 13-15) and NYHA functional classification (Tables 16-18). Table 13 summarizes all subjects in the OP population with new-onset HF. Table 14 summarizes data for men with new-onset HF, and Table 15 summarizes data for women with new-onset HF.

[0124] Table 16 shows the NYHA classification for the OP population with newly diagnosed HF. Table 17 shows men with newly diagnosed HF (NYHA functional classification), and Table 18 shows women with newly diagnosed HF.

[0125] [Table 13]

[0126] [Table 14]

[0127] [Table 15]

[0128] [Table 16]

[0129] [Table 17]

[0130] [Table 18]

[0131] OP group-receiver operating characteristic curve The receiver operating characteristic curve (ROC) shown in Figure 5 represents the clinical sensitivity and specificity for 185 registered subjects diagnosed with newly diagnosed HF and 848 subjects without HF. The area under the ROC curve for the NT-proBNP assay is 0.839, with a 95% confidence interval of 0.804–0.868.

[0132] Performance characteristics Measurement interval: 35–35,000 pg / mL (4.13–4130 pmol / L). The lower limit of the measurement interval is defined by the limit of quantification (LoQ). Results below the measurement interval are reported as less than 35 pg / mL (4.13 pmol / L).

[0133] Detection capability: Blank limit (LoB): 13 pg / ml (1.53 pmol / L) Limit of detection (LoD): 20 pg / ml (2.36 pmol / L) Limit of quantification (LoQ), 35pg / ml (4.13pmol / L) LoB and LoD values ​​are representative data. LoB corresponds to the highest measurement result, which is likely to be observed in a blank sample with a 95% probability. LoD corresponds to the lowest concentration of NT-proBNP that can be detected with a 95% probability. LoQ corresponds to the lowest amount of NT-proBNP in a sample for which the laboratory CV is 20%. Detection capability was determined according to CLSI document EP17-A2.

[0134] Clinical results A total of 3,128 subjects presenting with signs and symptoms of acute HF who visited an ED were prospectively enrolled in a multicenter clinical evaluation of one non-limiting embodiment of the NT-proBNP assay described herein. The diagnosis and severity of HF were determined by an independent central review committee of specialist clinicians (cardiologists). 1,148 subjects were confirmed to have acute HF, and 1,980 subjects were confirmed not to have HF.

[0135] Descriptive statistics for NT-proBNP test results (pg / ml) were determined for the ED population at the time of registration.

[0136] Table 19 summarizes the baseline characteristics for the entire ED population, while Table 20 summarizes the baseline characteristics of the ED population by age group.

[0137] [Table 19]

[0138] [Table 20-1] [Table 20-2]

[0139] likelihood ratio NT-proBNP results were stratified into three categories based on an overall rule-out cutoff value of 300 pg / ml and age-specific rule-in cutoff values, and compared to a confirmed diagnosis of acute heart failure (HF). The pre-test risk (or prevalence) of HF in this study was 36.7%. Compared to this pre-test risk, the risk of HF increased to 67.2% in subjects with a positive NT-proBNP result (post-test risk). The likelihood ratio compares the probability of an NT-proBNP test result due to acute heart failure to the probability of an NT-proBNP test result due to heart failure. For a positive NT-proBNP test result, a likelihood ratio of 3.53 indicates that individuals with acute HF are 3.5 times more likely to have a positive test result than those without acute HF. In subjects with inconclusive results, the post-test risk of HF decreased to 24.3%, and the probability of being in the HF population was almost half (LR 0.55) compared to the negative diagnosis population. In the case of a negative NT-proBNP test result, a likelihood ratio of 0.07 indicates that individuals with acute HF are 0.07 times (less than 1 / 10) more likely to test negative than individuals without acute HF.

[0140] A 2x3 contingency table format is used to analyze the clinical outcomes between the non-limiting embodiments of the NT-proBNP assay described herein and the confirmed diagnosis. Tables 21-32 summarize the comparison of NT-proBNP results and confirmed diagnoses for acute HF (positive or negative) using the following criteria: • Value greater than the age-specific rule-in cutoff value (positive) • Between the rule-in cutoff value and the rule-out cutoff value (uncertain) • Below the rule-out cutoff value (negative) LR should only be used for the ED population.

[0141] ED population – Clinical agreement between NT-proBNP results and acute HF diagnosis in non-limiting embodiments of the assays of this disclosure. Table 21 summarizes the results for all sex and age groups combined; Table 22 summarizes the results by age group; Table 23 summarizes the results for male subjects by age group; Table 24 summarizes the results for female subjects by age group. Analysis was also performed on relevant clinical subgroups of the ED population, as shown in Tables 25–32.

[0142] [Table 21]

[0143] [Table 22]

[0144] [Table 23]

[0145] [Table 24]

[0146] [Table 25]

[0147] [Table 26]

[0148] [Table 27]

[0149] [Table 28]

[0150] [Table 29]

[0151] [Table 30]

[0152] [Table 31]

[0153] [Table 32]

[0154] The following statements are based on results from clinical studies.

[0155] BMI of 30 kg / m² 2 The above patients have a BMI of 30 kg / m². 2 The false negative rate was higher compared to patients with a BMI of less than 30 kg / m². Of the total false negatives (44 / 1148), 41 (93%) had a BMI of 30 kg / m². 2 Of the patients mentioned above, one (2.2%) had a BMI of 30 kg / m². 2 The data originated from patients with a BMI of less than 10%, and two patients (4.4%) had an unknown BMI.

[0156] eGFR of 60 mL / min / 1.73 m 2 Patients with an eGFR of less than 60 mL / min / 1.73 m² 2 The false-positive rate was higher compared to patients with the above characteristics. The eGFR confirmed to be not acute HF was 60 mL / min / 1.73 m². 2 Of the 492 patients with a blood count below a certain level, 226 (45.9%) had NT-proBNP concentrations of ASC or higher. Those with an eGFR of 60 mL / min / 1.73 m² confirmed not to have acute HF. 2 Of the 1427 patients mentioned above, 251 (17.6%) had NT-proBNP concentrations above ASC. For the 61 patients (3.1%) who were confirmed not to have acute HF, eGFR was unknown.

[0157] Patients with a history of HF had a higher false-positive rate compared to patients without a history of HF. Of the 527 patients with a history of HF who were confirmed not to have acute HF, 243 (46.1%) had NT-proBNP concentrations of ASC or higher. Of the 1338 patients without a history of HF who were confirmed not to have HF, 216 (16.1%) had NT-proBNP concentrations of ASC or higher. Of the 1980 patients who were confirmed not to have acute HF, the history of HF was unknown for 115 (5.8%).

[0158] Variations in NT-proBNP concentration due to high BMI, low GFR, and a history of HF are widely supported by the literature.

[0159] Clinical outcomes in the OP population A total of 1033 patients with signs and symptoms of newly diagnosed HF were prospectively enrolled in a multicenter clinical evaluation of one non-limiting embodiment of the NT-proBNP assay described herein. Clinical outcomes were evaluated using a single cutoff value of 125 pg / ml.

[0160] The diagnosis and severity of HF were determined by an independent central review committee of specialist clinicians (cardiologists). 185 subjects were confirmed to have newly diagnosed HF, and 848 subjects were determined not to have HF.

[0161] [Table 33]

[0162] Clinical sensitivity and specificity The clinical sensitivity and specificity of the OP population and related OP clinical subgroups were determined by comparing the performance of non-limiting embodiments of the NT-proBNP assay of this disclosure with established diagnoses. A single cutoff of 126 pg / mL was used for established diagnoses of new-onset HF, and the analysis consisted of calculations of clinical sensitivity, clinical specificity, positive predictive value (PPV), and negative predictive value (NPV). The results are summarized in Tables 34-38.

[0163] Clinical sensitivity and specificity were determined according to CLSI document EP12-A2.

[0164] [Table 34]

[0165] [Table 35]

[0166] [Table 36]

[0167] [Table 37]

[0168] [Table 38]

[0169] accuracy The accuracy was determined according to CLSI document EP05-A3. Samples were assayed as two replicate tests over 20 days with two runs per day. The results in Table 39 represent the performance of the assay.

[0170] [Table 39-1] [Table 39-2]

[0171] reproducibility Reproducibility was determined according to CLSI document EP05-A3. Samples (N=90) were assayed at three facilities in three replicate tests, with two runs per day for five days. The results shown in Table 40 represent the performance of the assay.

[0172] [Table 40]

[0173] Equivalence of specimens Sample equivalence was determined using a Passing-Bablok regression model, in accordance with CLSI document EP09c-ed3. The degree of agreement between sample types may vary depending on the study design and the subject population.

[0174] [Table 41]

[0175] This assay was designed to have a correlation coefficient of 0.950–1.000, a slope of 1.00±0.05, and sections with a concentration of 6 pg / mL (0.708 pmol / L) or less.

[0176] interference Hemolysis, jaundice, and lipemia (HIL) Interference tests were performed according to CLSI document EP07-ed3. The substances listed in Table 42 do not interfere with the assay when present in serum at the indicated concentrations. The bias due to these substances does not exceed 10% at NT-proBNP concentrations of 123–137 pg / mL (14.5–16.2 ​​pmol / L) and 1612–1813 pg / mL (190–214 pmol / L).

[0177] [Table 42]

[0178] Other substances Interference tests were performed according to CLSI document EP07-ed3. The substances listed in Table 43 do not interfere with the assay when present in serum at the indicated concentrations. The bias due to these substances does not exceed 10% at NT-proBNP concentrations of 116–163 pg / mL (13.7–19.2 pmol / L) and 1478–2225 pg / mL (174–263 pmol / L).

[0179] [Table 43-1] [Table 43-2]

[0180] Cross-reactivity Cross-reactivity was determined according to CLSI document EP07-ed3. The cross-reactives listed in Table 44 were tested at NT-proBNP concentrations of 0 pg / mL (0 pmol / L) and 126–158 pg / mL (14.9–18.6 pmol / L).

[0181] [Table 44]

[0182] linearity Linearity testing was performed according to CLSI document EP06-ed2. The assay is linear over the measurement interval of 35–35,000 pg / mL (4.13–4130 pmol / L).

[0183] Onboard Dilution Recovery Serum samples were diluted using Multi-Diluent1 on the ADVIA Centaur XP System. The results in Table 45 represent the performance of the assay.

[0184] [Table 45]

[0185] High-dose hook effect High NT-proBNP concentrations can cause a paradoxical decrease in RLU (high-dose hook effect). In this assay, patient samples with NT-proBNP concentrations as high as 300,000 pg / mL (35,400 pmol / L), exceeding the measurement interval, showed values ​​above 35,000 pg / mL (4130 pmol / L).

[0186] standardization This assay is traceable to internal standards prepared using highly purified materials. Values ​​assigned to calibration standards are traceable to this standardization. Currently, there are no reference standards available for this assay. [Examples]

[0187] Tables 46-47 provide an overview of the features and reagents of a non-limiting embodiment of a kit constructed in accordance with this disclosure. The listed non-assay-related reagents (i.e., washing reagents, multi-purpose diluents, etc.) are available, for example, from Siemens Healthineers (Malvern, PA), but are not limited to these.

[0188] [Table 46]

[0189] [Table 47]

[0190] It should be noted that in some non-limiting embodiments, the Wright reagent, solid-phase reagent, auxiliary well reagent, calibration standard, multi-purpose diluent, and / or washing reagent can be stored unopened at 2–8°C. In addition, in some non-limiting embodiments, the Wright reagent, solid-phase reagent, and / or auxiliary well reagent are substantially stable for at least 36 days after being loaded. [Examples]

[0191] This embodiment includes a comparison of biotin interference observed between a prior art three-reagent NT-proBNP assay and a non-limiting embodiment of the two-reagent NT-proBNP assay of the present disclosure. As can be seen, the prior art three-reagent NT-proBNP assay showed biotin interference of -28% to -23% at a biotin concentration of 200 ng / mL. In contrast, even at biotin concentrations more than 17 times higher than those at which significant biotin interference was observed in the prior art assay, the assay of the present disclosure showed virtually no biotin interference (defined as a percentage bias of 10% or less).

[0192] [Table 48] [Examples]

[0193] This embodiment includes a comparison of biotin interference observed between a prior art three-reagent NT-proBNP assay and a non-limiting embodiment of the two-reagent NT-proBNP assay of the present disclosure. As can be seen, the acceptable percentage bias observed for the assay concentrations of the present invention occurs at biotin concentrations that are at least about 50 times higher than the biotin concentrations at which an acceptable percentage bias was observed in the prior art assay.

[0194]

Table 49

[0195] Non-limiting exemplary embodiments Exemplary embodiment 1. A kit for use in measuring the amount of NT-proBNP in a biological sample, comprising: (a) a solid-phase reagent comprising a solid support directly or indirectly conjugated with a first anti-NT-proBNP antibody or a binding fragment thereof; and (b) a light reagent comprising a second anti-NT-proBNP antibody or a binding fragment thereof labeled with an acridinium ester; wherein the first and second anti-NT-proBNP antibodies or binding fragments thereof bind to non-overlapping epitopes of NT-proBNP, thereby forming an immune complex comprising (a), (b) and NT-proBNP.

[0196] Exemplary embodiment 2. The kit according to exemplary embodiment 1, wherein the first anti-NT-proBNP antibody or a binding fragment thereof is biotinylated and at least a part of the surface of the solid support is coated with a biotin-specific binding molecule, whereby the first anti-NT-proBNP antibody or a binding fragment thereof is indirectly conjugated to the solid support.

[0197] Exemplary embodiment 3. The kit according to exemplary embodiment 2, wherein the biotin-specific binding molecule is selected from the group consisting of streptavidin, traptavidin and avidin.

[0198] Exemplary embodiment 4. The kit according to exemplary embodiment 3, wherein the biotin-specific binding molecule is streptavidin.

[0199] Exemplary embodiment 5. The kit according to any one of exemplary embodiments 1 to 4, wherein the solid support comprises magnetic particles and / or latex particles.

[0200] The kit according to any one of the exemplary embodiments 1 to 5, wherein the solid-phase reagent of exemplary embodiment 6(a) comprises a biotinylated monoclonal sheep anti-human antibody specific to NT-proBNP conjugated with magnetic particles whose surface is coated with streptavidin on at least a portion of it.

[0201] The kit according to any one of the exemplary embodiments 1 to 6, wherein the Wright reagent of exemplary embodiment 7(b) comprises an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP.

[0202] A kit according to any one of the exemplary embodiments 1 to 7, wherein exemplary embodiment 8(a) is packaged separately from (b).

[0203] Exemplary Embodiment 8A: A kit according to any one of Exemplary Embodiments 1 to 8, configured for use in an emergency department environment.

[0204] Exemplary Embodiment 8B. A kit according to any one of Exemplary Embodiments 1 to 8A, configured for use in an outpatient setting.

[0205] Exemplary Embodiment 8C: A kit according to any one of Exemplary Embodiments 1 to 8B, configured for use in an emergency department and / or outpatient setting.

[0206] Exemplary Embodiment 9. A method for determining the presence, severity and / or predisposition of heart failure (HF) in an individual, comprising: (a) a step of simultaneously, or all or part of sequentially, combining (i) a biological fluid sample; (ii) a solid-phase reagent comprising a solid support to which a first anti-NT-proBNP antibody or a binding fragment thereof is bound; and (iii) a Wright reagent comprising a second anti-NT-proBNP antibody or a binding fragment thereof labeled with an acridinium ester, wherein the first and second anti-NT-proBNP antibodies or their binding fragments bind to non-overlapping epitopes of NT-proBNP; (b) a step of incubating the reaction mixture under conditions that enable (ii) and (iii) to bind to NT-proBNP present in the biological fluid sample to form an immune complex; and (c) a step of measuring the amount of the formed immune complex to obtain a measurement of NT-proBNP in the sample.

[0207] Exemplary Embodiment 10.(d) The method according to Exemplary Embodiment 9, further comprising the step of obtaining an HF score based on a measurement of NT-proBNP in a sample using a mathematical algorithm.

[0208] Exemplary Embodiment 11. The method according to Exemplary Embodiment 9 or 10, wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, and combinations thereof.

[0209] Exemplary Embodiment 12. The method according to any one of Exemplary Embodiments 9 to 11, wherein the first anti-NT-proBNP antibody or its binding fragment is biotinylated, and at least a portion of the surface of a solid support is coated with a biotin-specific binding molecule, thereby indirectly binding the first anti-NT-proBNP antibody or its binding fragment to the solid support.

[0210] Exemplary Embodiment 13. The method according to Exemplary Embodiment 12, wherein the biotin-specific binding molecule is selected from the group consisting of streptavidin, traptabidine, and avidin.

[0211] Exemplary Embodiment 13A. The method according to Exemplary Embodiment 13, wherein the biotin-specific binding molecule is streptavidin.

[0212] Exemplary Embodiment 14. The method according to any one of Exemplary Embodiments 9 to 13A, wherein the solid support comprises magnetic particles and / or latex particles.

[0213] Exemplary Embodiment 15. The solid phase reagent of (ii) comprises a biotinylated monoclonal sheep anti-human antibody specific for NT-proBNP conjugated with magnetic particles at least a part of the surface of which is coated with streptavidin, the method according to any one of Exemplary Embodiments 9 to 14.

[0214] Exemplary Embodiment 16. The light reagent of (iii) comprises an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab’)2 fragment specific for NT-proBNP, the method according to any one of Exemplary Embodiments 9 to 15.

[0215] Exemplary Embodiment 17. The method according to any one of Exemplary Embodiments 9 to 16, wherein substantially no interference from biotin present in the biological fluid sample is observed.

[0216] Exemplary Embodiment 17A. The method according to Exemplary Embodiment 17, wherein substantially no biotin interference is defined as a percent bias of 10% or less.

[0217] Exemplary Embodiment 17B. The method according to Exemplary Embodiment 17 or Exemplary Embodiment 17A, wherein substantially no biotin interference is observed at a biotin concentration in the biological fluid sample up to about 3510 ng / ml.

[0218] Exemplary Embodiment 18. The method according to any one of Exemplary Embodiments 9 to 17, wherein at least one of steps (b), (c) and (d) is performed by an automatic analyzer. [[ID=二十九]]

[0219] Exemplary Embodiment 19. The method according to Exemplary Embodiment 18, wherein step (c) is further defined as a step of measuring the amount of relative luminescence units (RLUs), the amount of RLUs being directly proportional to the amount of NT-proBNP in the sample.

[0220] Exemplary Embodiment 19A. The method according to any one of Embodiments 9 to 19, performed in an emergency department setting.

[0221] Exemplary Embodiment 19B. The method according to any one of Exemplary Embodiments 9 to 19A, wherein the method or a portion thereof is performed in an outpatient setting. For example, a sample may be obtained in an outpatient setting or from an outpatient patient population and shared with a facility suitable for performing the method of the Disclosure or for using the kit of the Disclosure. In some embodiments, sharing may be carried out by sending the sample to the facility under conditions suitable for transporting biomaterials.

[0222] Exemplary Embodiment 19C. The method according to any one of Exemplary Embodiments 9 to 19B, performed in an emergency department, point of care, and / or outpatient setting.

[0223] Exemplary Embodiment 20. A non-temporary computer-readable medium containing an executable instruction that, if executed, causes a processor to perform an operation including the method described in any one of the Exemplary Embodiments 9 to 19C.

[0224] Exemplary Embodiment 21. A kit for use in measuring the amount of NT-proBNP in a biological sample, comprising: (a) a solid-phase reagent comprising a biotinylated monoclonal sheep anti-human antibody specific to NT-proBNP conjugated with magnetic particles whose surface is coated with streptavidin on at least a portion of it; and (b) a Wright reagent comprising an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP, wherein the antibody from (a) and the F(ab')2 fragment from (b) bind to non-overlapping epitopes of NT-proBNP, thereby forming an immunocomplex comprising (a), (b), and NT-proBNP.

[0225] Exemplary Embodiment 22. The kit according to Exemplary Embodiment 21, wherein (a) is packaged separately from (b).

[0226] Exemplary Embodiment 22A: The kit according to Exemplary Embodiment 21 or 22, configured for use in an emergency departmental environment.

[0227] Exemplary Embodiment 22B: A kit according to any one of the exemplary embodiments 21-22A, configured for use in an outpatient setting.

[0228] Exemplary Embodiment 23. A method for determining the presence, severity and / or predisposition of heart failure (HF) in an individual, comprising: (a) a step of simultaneously, or all or part of sequentially, combining the Wright reagent, which comprises (i) a biological fluid sample; (ii) a biotinylated monoclonal sheep anti-human antibody specific to NT-proBNP conjugated with magnetic particles having at least a portion of their surface coated with streptavidin; and (iii) a Wright reagent containing an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP, wherein the antibody from (ii) and the F(ab')2 fragment from (iii) bind to non-overlapping epitopes of NT-proBNP; and (b) incubating the reaction mixture under conditions that enable (ii) and (iii) to bind to NT-proBNP present in the biological fluid sample to form an immune complex; and (c) measuring the amount of the formed immune complex to obtain a measurement of NT-proBNP in the sample.

[0229] Exemplary Embodiment 24.(d) The method according to Exemplary Embodiment 23, further comprising the step of obtaining an HF score based on a measurement of NT-proBNP in a sample using a mathematical algorithm.

[0230] Exemplary Embodiment 25. The method according to Exemplary Embodiment 23 or 24, wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, and combinations thereof.

[0231] Exemplary Embodiment 26. The method according to any one of the exemplary embodiments 23 to 25, wherein substantially no interference from biotin present in the biological fluid sample is observed.

[0232] Exemplary Embodiment 26A. The method according to Exemplary Embodiment 26, wherein substantially no biotin interference is defined as a percentage bias of 10% or less.

[0233] Exemplary Embodiment 26B: The method according to Exemplary Embodiment 26 or 26A, wherein biotin interference is substantially not observed at biotin concentrations in biological fluid samples up to approximately 3510 ng / ml.

[0234] Exemplary Embodiment 27. The method according to any one of the exemplary embodiments 23 to 26, wherein at least one of steps (b), (c), and (d) is performed in an automated analyzer.

[0235] Exemplary Embodiment 28. The method according to Exemplary Embodiment 27, wherein step (c) is further defined as a step of measuring the amount of relative luminescence units (RLUs), the amount of RLUs being directly proportional to the amount of NT-proBNP in the sample.

[0236] Exemplary Embodiment 28A. The method according to any one of the exemplary embodiments 23 to 28, performed in an emergency department setting.

[0237] Exemplary Embodiment 28B. The method according to any one of the exemplary embodiments 23 to 28A, performed in an outpatient setting.

[0238] Exemplary Embodiment 28C. The method according to any one of the exemplary embodiments 23-28, performed in an emergency department and / or outpatient setting.

[0239] Exemplary Embodiment 29. A non-temporary computer-readable medium containing an executable instruction that, if executed, causes a processor to perform an operation including the method described in any one of the Exemplary Embodiments 23 to 28C.

[0240] Exemplary Embodiment 30. A method for determining the presence, severity, and / or predisposition of heart failure (HF) in an individual, (a)(i) biological fluid samples; (ii) A solid-phase reagent comprising a solid support to which a first anti-NT-proBNP antibody or a binding fragment thereof is bound; and (iii) A Wright reagent comprising a second anti-NT-proBNP antibody labeled with acridinium ester or a binding fragment thereof, wherein the first and second anti-NT-proBNP antibodies or binding fragments bind to non-overlapping epitopes of NT-proBNP. A process of combining these simultaneously, or all or part of them sequentially, to form a reaction mixture; (b) a step of incubating the reaction mixture, (ii) and (iii) under conditions that enable them to bind to NT-proBNPs present in the biological fluid sample and form an immunocomplex; and (c) A step to measure the amount of formed immune complexes and obtain a measurement value for NT-proBNP in the sample. Includes, Depending on the case, the following steps may be taken: (d) A step of obtaining an HF score based on measured values ​​of NT-proBNP in a sample using a mathematical algorithm, wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, and combinations thereof. A method that includes this.

[0241] Exemplary Embodiment 31. Embodiment 30, wherein the method is performed in an emergency department, point of care, and / or outpatient setting.

[0242] Exemplary Embodiment 32. Embodiments 30-31, in which a biological fluid sample is obtained in an outpatient clinical setting and subsequently added to the reaction mixture. It will be provided.

[0243] Exemplary Embodiment 33. Embodiments 30-32, characterized in that the individual originates from the outpatient hierarchy.

[0244] Exemplary Embodiment 34. Embodiments 30-33, wherein a first anti-NT-proBNP antibody or its binding fragment is biotinylated, and at least a portion of the surface of a solid support is coated with streptavidin, thereby indirectly binding the first anti-NT-proBNP antibody or its binding fragment to the solid support, the solid support comprising magnetic particles and / or latex particles, the solid-phase reagent of (ii) comprising a biotinylated monoclonal sheep anti-human antibody specific to NT-proBNP conjugated with magnetic particles whose surface at least a portion is coated with streptavidin; and the Wright reagent of (iii) comprising an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP, no biotin interference is observed at biotin concentrations in the biological fluid sample up to about 3510 ng / ml, and at least one of steps (b), (c), and (d) is performed using an automated analyzer.

[0245] Accordingly, the present disclosure provides compositions, kits, systems and / or methods that fully satisfy the above-mentioned objectives and advantages. While the present disclosure is described in conjunction with the specific drawings, experiments, results and representations described above, it is obvious that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to encompass all such alternatives, modifications and variations that are in the spirit and broad scope of the present disclosure. [Explanation of Symbols]

[0246] 1100 Computer System 1110 processor 1120 memory 1130 Storage device 1140 Input / Output Devices 1150 System Bus 1160 Analysis Machine

Claims

1. A kit for determining the amount of NT-proBNP in a biological sample, (a) A solid-phase reagent comprising a solid support to which a first anti-NT-proBNP antibody or a binding fragment thereof is directly or indirectly bound; and (b) Wright reagent containing a second anti-NT-proBNP antibody labeled with acridinium ester or a binding fragment thereof; Includes, The first and second anti-NT-proBNP antibodies or their binding fragments bind to non-overlapping epitopes of NT-proBNP, thereby forming an immune complex comprising (a), (b), and NT-proBNP. kit.

2. The kit according to claim 1, wherein the first anti-NT-proBNP antibody or its binding fragment is biotinylated, and at least a portion of the surface of the solid support is coated with streptavidin, thereby indirectly binding the first anti-NT-proBNP antibody or its binding fragment to the solid support.

3. The kit according to claim 1, wherein the solid support comprises magnetic particles and / or latex particles.

4. The solid-phase reagent of (a) comprises a biotinylated monoclonal sheep anti-human antibody specific to NT-proBNP conjugated with magnetic particles whose surface is coated with streptavidin on at least a portion of it; and (b) The Wright reagent contains an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP. The kit according to claim 1.

5. (a) is packaged separately from (b), the kit according to claim 1.

6. The kit according to claim 1, configured for use in an emergency department and / or outpatient setting.

7. A method for determining the presence, severity, and / or predisposition of heart failure (HF) in an individual, (a)(i) biological fluid samples; (ii) A solid-phase reagent comprising a solid support to which a first anti-NT-proBNP antibody or a binding fragment thereof is bound; and (iii) A Wright reagent comprising a second anti-NT-proBNP antibody labeled with acridinium ester or a binding fragment thereof, wherein the first and second anti-NT-proBNP antibodies or binding fragments bind to non-overlapping epitopes of NT-proBNP. A process of combining these simultaneously, or all or part of them sequentially, to form a reaction mixture; (b) Incubating the reaction mixture under conditions that enable (ii) and (iii) to bind to NT-proBNPs present in the biological fluid sample to form an immune complex; and (c) A step of measuring the amount of formed immune complexes to obtain a measurement value of NT-proBNP in the sample. A method that includes this.

8. (d) A step of obtaining an HF score based on measured values ​​of NT-proBNP in the sample using a mathematical algorithm. The method according to claim 7, further comprising:

9. The method according to claim 7, wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, and combinations thereof.

10. The method according to claim 7, wherein the first anti-NT-proBNP antibody or its binding fragment is biotinylated, and at least a portion of the surface of the solid support is coated with streptavidin, thereby indirectly binding the first anti-NT-proBNP antibody or its binding fragment to the solid support.

11. The method according to claim 7, wherein the solid support comprises magnetic particles and / or latex particles.

12. The solid-phase reagent of (ii) comprises a biotinylated monoclonal sheep anti-human antibody specific to NT-proBNP conjugated with magnetic particles whose surface is coated with streptavidin on at least a portion of it; and The (iii) Wright reagent contains an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP. The method according to claim 7.

13. The method according to claim 7, wherein no biotin interference is observed at biotin concentrations in biological fluid samples up to approximately 3510 ng / ml.

14. The method according to claim 7, wherein at least one of steps (b), (c), and (d) is performed using an automated analyzer.

15. The method according to claim 14, wherein step (c) is further defined as a step of measuring the amount of relative luminescence units (RLU), the amount of RLU being directly proportional to the amount of NT-proBNP in the sample.

16. The method according to claim 7, performed in an emergency department, point of care, and / or outpatient setting.

17. The method according to claim 7, wherein the biological fluid sample is obtained in an outpatient clinical setting and subsequently added to the reaction mixture.

18. The method according to claim 7, wherein the individual is characterized by being of outpatient origin.

17. A non-temporary computer-readable medium including an executable instruction that, if executed, causes a processor to perform an operation including the method according to claim 7.

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