Methods, reagents and kits for modulating immunoassay signals

Abstract

Methods, assays, systems, and kits for determining the presence, severity, and / or susceptibility of heart failure (HF) in an individual are disclosed. The methods utilize a solid matrix in which streptavidin-coated particles are pre-incubated with a biotinylated antibody to form a solid phase reagent. Since the solid phase reagent is now present in a pre-complex form with the biotinylated antibody, there is less opportunity for biotin from the sample to interfere with the assay. The methods, systems, assays, and kits of the present disclosure allow NT-proBNP assays to demonstrate no biotin interference at concentrations of up to 3510 ng / mL biotin.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 514,108, filed July 17, 2023. The entire contents of the aforementioned patent application are hereby incorporated by reference. Invention Field

[0002] This disclosure pertains to the field of immunoassays and assay formats suitable for modulating signals by altering their signal-to-noise ratio. Furthermore, this disclosure relates to stabilizing reagents and eliminating interference from free biotin in biological samples. In some non-limiting embodiments, this disclosure modulates the signal-to-noise ratio of NT-proBNP sandwich immunoassays suitable for high-throughput analyzers. Background of the Invention Left ventricular dysfunction can occur as part of coronary artery disease, hypertension, valvular heart disease, and primary cardiomyopathy. If left ventricular dysfunction remains untreated and progresses, the potential for mortality is high, for example, due to sudden cardiac death. Chronic heart failure is a clinical syndrome caused by impaired cardiac pumping function. Based on symptoms, the presence and severity of heart failure can be classified as stages I-IV by the New York Heart Association (NYHA), for example. Clinical tests and imaging procedures 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 the control of cardiovascular function has been established. Brain natriuretic peptide biomarkers are commonly used to aid in the diagnosis and assessment of the severity of heart failure (HF) in patients.

[0005] ANP and BNP have natriuretic and diuretic properties. As antagonists of the renin-angiotensin-aldosterone system, ANP and BNP affect electrolyte and fluid balance in the body. In subjects with left ventricular dysfunction, serum and plasma concentrations of BNP are increased, as are concentrations of the biologically inactive pro-bNP. ProBNP consists of 108 amino acids. It is primarily secreted by the left ventricle of the heart and, in the process, is cleaved into physiologically active BNP (amino acids 77–108) and the N-terminal fragment NT-proBNP (amino acids 1–76) (see, for example, 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).

[0006] Biomarkers such as NT-proBNP serve as surrogates for clinically meaningful outcomes and may or may not reflect the underlying pathogenesis of a disease. Examples of clinical utility include diagnosis, prediction of disease progression or regression, and prediction of mortality. Biomarkers should be readily available, reliably measurable, and usable for continuous monitoring. Ideally, they should also offer the advantages of currently used clinical measurements in terms of ease of use, time frame, and / or cost.

[0007] Certain substances in biological samples problematically interfere with assay formats used to measure biomarkers. It is well known that the presence of biotin in biological samples can bias assay results and / or interfere with assays, for example, in assays where a biotinylated antibody is a reagent separate from a solid phase comprising biotin-binding molecules, such as (but not limited to) unconjugated streptavidin-coated particles. Assay formats problematically allow time for any naturally occurring biotin in the biological sample to recombine with streptavidin-coated particles, resulting in interference where the biotinylated antibody is blocked from binding to the streptavidin-coated particles.

[0008] Existing technologies of interest include those granted to Janzen et al., named Method and Composition For Stabilizing Liquid Reagents U.S. Patent No. 8,252,605 (incorporated herein by reference in its entirety). However, the shortcoming of the prior art is that, particularly for the NT-proBNP detection immunoassay according to this disclosure, it does not show the improvement in reducing biotin interference as shown and described herein.

[0009] Therefore, there is a persistent need in the art for improved assays, such as NT-proBNP assays with improved performance, where problematic free or naturally occurring biotin is present or can be present in biological samples at concentrations sufficient to cause interference. This disclosure relates to such biomarker assay formats, and compositions / reagents / kits containing reagents for measuring biomarkers, together with methods of using the same. Brief description of the attached diagram The embodiments of this disclosure, which have been briefly summarized above and discussed in more detail below, can be understood by referring to the exemplary embodiments of this disclosure depicted in the accompanying drawings. However, the drawings only illustrate typical embodiments of this disclosure and are therefore not to be considered as limiting the scope, as other equally effective embodiments of this disclosure may be employed.

[0011] Figure 1A prior art immunoassay for NT-proBNP is described. The prior art immunoassay utilizes three reagents: streptoacid-coated particles (reagent 1), a biotinylated antibody (reagent 2), and an acridinium ester-labeled antibody (reagent 3). Two antibodies bind to non-overlapping epitopes of NT-proBNP, allowing the formation of a sandwich complex (product 1).

[0012] Figure 2 The effects of biotin interference on Figure 1 The role of existing immunoassay techniques.

[0013] Figure 3 A non-limiting embodiment of the NT-proBNP immunoassay constructed according to this disclosure is described. In this embodiment, a solid-phase reagent is produced by pre-incubating streptavidin-coated particles with a biotinylated antibody (or a binding fragment thereof) prior to sample addition. Because the solid-phase reagent is present with the biotinylated antibody in a pre-complexed form, biotin interference from free or native biotin in the sample is eliminated or substantially eliminated during the immunoassay.

[0014] Figure 4 An exemplary block diagram is depicted of a computer system 1100 suitable for performing the methods of this disclosure on a chemical analyzer.

[0015] Figure 5 The recipient operating characteristic (ROC) curves are shown graphically, which confirm the clinical sensitivity and specificity of the immunoassay constructed according to this disclosure.

[0016] For ease of understanding, the same reference numerals have been used to denote common elements in the accompanying drawings, where possible. The drawings are not drawn to scale and may be simplified for clarity. Elements and features of one embodiment may be beneficially incorporated into other embodiments without further description. Invention Details Before explaining in detail at least one embodiment of this disclosure through exemplary language and results, it should be understood that this disclosure is not limited in its application to the construction details and component arrangements set forth in the description below. This disclosure can have other embodiments, or can be practiced or carried out in various ways. Therefore, the language used herein is intended to give the broadest possible scope and meaning; and the embodiments are intended to be exemplary—not exhaustive. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered restrictive.

[0018] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure should have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms should include plural terms, and plural terms should include singular terms. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and described in the various general and more specific references cited and discussed throughout this specification. The nomenclature, laboratory procedures, and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well known and commonly used in the art.

[0019] All patents, published patent applications, and non-patent publications mentioned in this specification indicate the level of skill of a person skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, to the extent that each individual patent or publication specifically and individually indicates its inclusion by reference.

[0020] According to this disclosure, all compositions, devices, kits, and / or methods disclosed herein can be prepared and performed without excessive experimentation. While compositions, devices, kits, and / or methods have been described with reference to specific embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions, devices, kits, and / or methods described herein, as well as the steps or sequence of steps of the methods, without departing from the concept, spirit, and scope of this disclosure. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0021] definition Unless otherwise stated, the following terms shall be understood to have the following meanings as used in accordance with this disclosure: When used in conjunction with the term "comprising" in the claims and / or specification, the term "a" or "an" may mean "one / a," but it is also consistent with the meaning of "one or more / a combination of," "at least one / a," and "one / a or more than one / a." Therefore, unless the context clearly indicates otherwise, the terms "a," "an," and "the / described" include plural indicators. Thus, for example, reference to "compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "a plurality" means "two or more."

[0022] The term "at least one" should be understood to include any quantity of one or more, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term it is attached to; furthermore, a quantity of 100 / 1000 is not considered limiting, as higher limits can also produce satisfactory results. Additionally, the term "at least one of X, Y, and Z" should be understood to include individual X, individual Y, and individual Z, as well as any combination of X, Y, and Z.

[0023] For example, the use of ordinal numbers (i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for the purpose of distinguishing two or more items and, unless otherwise expressly stated, is not intended to imply any order or sequence or importance of one item over another or any additional order.

[0024] The use of the term "or" in the claims is intended to mean inclusive "and / or" unless it is explicitly stated 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).

[0025] As used herein, any reference to “one embodiment,” “an embodiment,” “some embodiments,” “one example,” “for example,” or “an example” means that a particular element, feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. For example, the phrases “some embodiments” or “one example” appearing throughout the specification do not necessarily refer to the same embodiment. Furthermore, all references to one or more embodiments or examples are construed as non-limiting to the claims.

[0026] Throughout this application, the term "about" is used to indicate values ​​including inherent error variations in the composition / instrument / device, variations in the method used to determine the value, or variations present in the subject of study. For example, but not as a limitation, when using the term "about," the specified value can vary from a specified value by positive or negative 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 suitable for performing the disclosed methods and as understood by one of ordinary skill in the art.

[0027] The term "antibody" is used herein in its broadest sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and their conjugates (e.g., but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, biantibodies, single-chain antibodies, and other antibody fragments and their conjugates that retain at least a portion of the variable region of an intact antibody), antibody substitute proteins or peptides (i.e., modified binding proteins / peptides), and combinations or derivatives thereof. Antibodies can 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).

[0028] Consistent with its use in the art, the term "biomarker" or "biological marker" is used herein to refer to an entity whose presence, level, or form is associated with a particular biological event or state of interest, such that it is considered a "marker" of that event or state. To name just a few examples, in some non-limiting embodiments, a biomarker may be or include markers of a particular disease state, or markers of the likelihood that a particular disease, symptom, or condition may develop, occur, or recur. In some non-limiting embodiments, a biomarker may be or include markers of a particular disease or treatment outcome, or the likelihood thereof. Thus, in some non-limiting embodiments, a biomarker predicts a relevant biological event or state of interest; in some non-limiting embodiments, a biomarker foreshadows a relevant biological event or state of interest; and in some non-limiting embodiments, a biomarker diagnoses a relevant biological event or state of interest. In some non-limiting embodiments, a biomarker is a possible biomarker of a relevant biological event or state of interest. A biomarker can be an entity of any chemical class. For example, in some non-limiting embodiments, a biomarker may be or include nucleic acids, peptides, small molecules, or combinations thereof. 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 generated or present in extracellular fluids such as blood, urine, tears, saliva, cerebrospinal fluid, etc.).

[0029] As described herein, in some non-limiting embodiments, the biomarker is an NT-proBNP biomarker. As used herein, “NT-proBNP biomarker” refers to a biomarker for heart failure (HF). In some non-limiting embodiments, one or more NT-proBNP biomarkers comprise the N-terminal fragment of proBNP, NT-proBNP (amino acids 1-76). (See, for example, 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).

[0030] The term "characteristic fragment" refers to a fragment of a biomarker (e.g., the NT-proBNP biomarker) sufficient to identify the biomarker from which the fragment is derived. For example, in some non-limiting embodiments, a "characteristic fragment" of a biomarker is a characteristic fragment containing an amino acid sequence or set of amino acid sequences that together allow the biomarker from which the fragment is derived to be distinguished from other possible biomarkers, proteins, or peptides. In some non-limiting embodiments, the characteristic fragment comprises at least 10, at least 20, at least 30, at least 40, or at least 50 amino acids. In some non-limiting embodiments, a characteristic fragment refers to a fragment of a biomarker that has at least 90%, at least 95%, or at least 99% sequence identity with the biomarker from which the characteristic fragment is derived.

[0031] The term "hybridization" refers to the physical property of annealing a single-stranded nucleic acid molecule (e.g., DNA or RNA) to a complementary nucleic acid molecule. Hybridization can generally be evaluated in a variety of contexts—including in which interacting nucleic acid molecules are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise bound to a vector entity and / or in a biological system or cell). In some non-limiting embodiments, hybridization can be detected by hybridization techniques, such as those selected from in situ hybridization (ISH), microarrays, RNA blotting, DNA blotting, etc. 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%, at least 70% of the single-stranded nucleic acid molecules are annealed to the complementary nucleic acid molecule). Hybridization techniques and methods used to evaluate hybridization are well known in the art. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Press, Plainview, NY. Those skilled in the art will understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least the desired level of complementarity will hybridize stably, while sequences with lower complementarity will not. For examples of hybridization conditions and parameters, see, for instance, Sambrook et al., 1989, *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al., 1994, *Current Protocols in Molecular Biology*, John Wiley & Sons, Seculus, NJ.

[0032] As used herein, the term "detection reagent" refers to any detectable element, molecule, functional group, compound, fragment, or part. In some non-limiting embodiments, the detection reagent is provided or used alone. In some non-limiting embodiments, the detection reagent is provided and / or used in combination (e.g., linking) with another reagent. Examples of detection reagents 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, 123I, 64 Cu、 187 Re、 111 In、 90 Y、 99m Tc, 177 Lu、 89 Zr, fluorescent dyes, chemiluminescent agents (e.g., acridinium esters, stable dioxanes, 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, colloidal gold, etc.), biotin, digitalisin, haptens, and proteins for which antiserum or monoclonal antibodies are available.

[0033] As used herein, the term “diagnostic test” refers to a step or series of steps performed or performed to obtain information that can be used to determine whether a patient has a disease, symptom, or condition and / or classify the disease, symptom, or condition into a phenotypic category or be significant for the prognosis of the disease, symptom, or condition, or be likely to respond to treatment (general treatment or any particular treatment) for the disease, symptom, or condition. Similarly, the term “diagnosis” refers to providing any type of diagnostic information, including but not limited to whether a subject is likely to have or develop a disease, symptom, or condition; the state, stage, or characteristics of the disease, symptom, or condition manifested in the subject; information relating to the nature or classification of a condition such as a tumor or cardiac condition; information relating to prognosis; and / or information useful in selecting appropriate treatment or additional diagnostic tests. Treatment selection may include the selection of a specific therapeutic agent or other treatment modality such as surgery, radiation, etc.; the choice of whether to stop or deliver treatment; choices related to the dosing regimen (e.g., the frequency or level of one or more doses of a specific therapeutic agent or combination of therapeutic agents); and so on. The selection of additional diagnostic tests may include more specific tests for a given disease, symptom, or condition.

[0034] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “contain” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional elements or method steps not listed. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to those elements but may include other elements not expressly listed or inherently present therein.

[0035] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if the order is significant in the particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so on. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limitation on the number of items or terms in any combination.

[0036] As used herein, the term "substantially" means that the event or situation subsequently described occurs completely, or occurs to a large extent or to a large degree. For example, when relating to a particular event or situation, the term "substantially" means that the event or situation subsequently described occurs at least 80% of the time, or at least 85% of the time, at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are within close proximity to each other but not 100% adjacent, or that a portion of one item is not 100% adjacent to the other item but is within close proximity to the other item.

[0037] As used herein, the phrase “related to” includes both direct and indirect relationships between two parts. Non-limiting examples of relatedness include covalent bonding of one part to another via a direct bond or via a spacer group; non-covalent bonding of one part to another directly or by means of a specific binding pair member of the part; and incorporation of one part into another, for example, by dissolving one part into another or by synthesis and coating one part onto another.

[0038] As used herein, the term "biofluid sample" should be understood to include any liquid test sample that can be obtained from a patient and is utilized in accordance with this disclosure. Examples of biofluid samples that can be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), serum, EDTA plasma, lithium heparin plasma, combinations thereof, etc.

[0039] As used herein, when the term “volume” refers to a liquid test sample utilized according to this disclosure, it generally refers to the volume of a liquid test sample in the range of about 0.1 µl to about 100 µl, or about 1 µl to about 75 µl, or about 2 µl to about 60 µl, or less than or equal to about 50 µl.

[0040] As used herein, the term "patient" includes both humans and veterinary subjects. In some non-limiting embodiments, the patient is a mammal. In some other non-limiting embodiments, the patient is a human. For diagnostic / therapeutic purposes, the term "mammal" means any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo animals, sporting animals, or pet animals such as dogs, horses, cats, cattle, etc.

[0041] "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 medicine for treating a particular disease, symptom, or condition, a 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.

[0042] "Point-of-care testing" refers to diagnostic tests performed in real time, allowing for rapid completion of the test compared to comparable tests without such a system. Point-of-care testing can be performed quickly and on-site, such as in a doctor's office, at the bedside, in a statistical laboratory, emergency room, or other such locations, particularly where rapid and accurate results are required. Patient presence is possible but not mandatory. Point-of-care includes, but is not limited to, emergency rooms, operating rooms, hospital laboratories and other clinical laboratories, doctor's offices, on-site settings, or any situation where rapid and accurate results are expected.

[0043] As used herein specifically (but not as a limitation) in the terms “target analyte-specific binding coupler” or “biotin-specific binding coupler,” the term “specific binding coupler” should be understood to mean any molecule capable of specifically binding to a target analyte. For example, but not as a limitation, a binding coupler can be an antibody, receptor, ligand, aptamer, molecularly imprinted polymer (i.e., an inorganic matrix), combinations or derivatives thereof, and any other molecule capable of specifically binding to a target analyte.

[0044] As used herein, the term "immunoassay" refers to a assay that determines the presence of a diagnostic biomarker in a biological sample by reacting the sample with an antibody (or a fragment thereof) that specifically binds to a diagnostic biomarker or a characteristic fragment thereof, wherein the reaction is performed under conditions and at a time that allow for the formation of an immune complex between the antibody (or a fragment thereof) and the diagnostic biomarker. Quantification of such immune complexes is then performed.

[0045] Sample: As used herein, the term "sample" refers to a biological sample obtained from or derived from a human subject, as described herein. In some embodiments, the biological sample includes biological tissue or fluid. In some embodiments, the biological sample may include blood; blood cells; tissue or fine-needle biopsy samples; body fluids containing cells; free-floating nucleic acids; cerebrospinal fluid; lymph; tissue biopsy samples; surgical samples; other body fluids, secretions and / or excretions; and / or cells derived therefrom. In some embodiments, the biological sample includes cells obtained from an individual, such as a human or animal subject. In some embodiments, the obtained cells are or include cells from the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, or body fluid (e.g., blood) collection. In some embodiments, the sample is cardiac tissue obtained from a subject. In some implementations, as will be clear 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 reagents thereto). For example, semi-permeable membrane filtration is used. As another example of sample processing, the sample may be a plasma sample treated with an anticoagulant selected from EDTA, heparin, and citrate. As another example of sample processing, the sample may be processed to isolate one or more proteins (e.g., by capturing proteins with one or more antibodies). "Processed sample" may include, for example, nucleic acids or peptides extracted from the sample or obtained by subjecting the primary sample to techniques such as mRNA amplification or reverse transcription, isolation and / or purification of certain components.

[0046] Subject: As used herein, the term “subject” refers to a biological entity, such as a mammal (e.g., a human). In some embodiments, a human subject is an adult, adolescent, or pediatric 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 has a disease, condition, or status, such as a disease, condition, or status that can be treated as provided herein. In some embodiments, a subject is susceptible to a disease, condition, or status; in some embodiments, susceptible subjects are susceptible to a disease, condition, or status and / or show an increased risk of developing a disease, condition, or status (compared to the average risk observed in a reference subject or population). In some embodiments, a subject exhibits one or more symptoms of a disease, condition, or status. In some embodiments, a subject does not exhibit a specific symptom (e.g., clinical manifestations of a disease) or characteristic of a disease, condition, or status. In some embodiments, a subject does not exhibit any symptoms or characteristics of a disease, condition, or status such as heart failure. In some embodiments, a subject is a patient. In some implementations, the subject is an individual to whom a diagnosis and / or treatment has been administered and / or administered.

[0047] Threshold: As used herein, the term "threshold" refers to a value (or values) used as a reference for obtaining information about a measurement result and / or classifying the measurement result, such as a measurement result obtained in an assay. The threshold may be determined based on one or more control samples. The threshold may be determined before, during, or after the measurement of interest. In some embodiments, the threshold may be a series of values. In some embodiments, the threshold may be a value (or a series of values) reported in a relevant domain (e.g., values ​​found in a standard table).

[0048] The term "stratification" refers to dividing a potential patient group or patient group into subgroups, such as tiers or blocks. In the implementation, a "tier" refers to a specific portion of a patient population.

[0049] Turning now to the various non-limiting embodiments of this disclosure. This document describes improved biomarker assay formats, and compositions / reagents / kits containing reagents for measuring biomarkers, along with methods of using them. In some non-limiting embodiments, this disclosure provides improved assays, such as (but not limited to) improved NT-proBNP assays, which exhibit improved performance in cases where problematic free or naturally occurring biotin is present or may be present in biological samples and could cause interference in existing assay formats. In embodiments, the biomarker assay format is formulated to modulate the signal from the signal portion.

[0050] In some non-limiting embodiments, the assay comprises a solid support (e.g., but not limited to one or more bead substrates or particles) coated with one or more streptavidin proteins, pre-incubated with one or more biotinylated antibodies (or their binding fragments) to form one or more solid-phase reagents suitable for the immunoassay, while eliminating or substantially eliminating interference from free biotin in the biological sample. In some non-limiting embodiments, this disclosure adjusts the signal-to-noise ratio for NT-proBNP sandwich immunoassays suitable for high-throughput analyzers. In some non-limiting embodiments, because the solid-phase reagent is present with the biotinylated antibody in a pre-composite form, there is substantially no opportunity for interference from free or native biotin from the sample. In some non-limiting embodiments, for example for NT-proBNP analytes, "no interference with biotin" is defined as a percentage deviation of ≤10%. In some non-limiting embodiments, this disclosure allows NT-proBNP assays to claim no interference up to 3510 ng / mL of biotin.

[0051] In some non-limiting embodiments, this immunoassay can be run on a high-throughput chemistry analyzer capable of performing more than 20, 30, 40, 50, 75, 100 or more tests per hour. In some non-limiting embodiments, this disclosure also includes a non-transient computer-readable medium suitable for performing the methods of this disclosure on a chemistry analyzer.

[0052] In certain specific (but non-limiting) embodiments, the immunoassay may use a labeled second antibody that is also bound to a serum marker or a fragment thereof to detect a complex between the serum marker or a characteristic fragment thereof and the serum marker-binding antibody. In certain non-limiting embodiments, a sandwich immunoassay is used, wherein the serum marker-binding antibody may be a capture antibody attached to an insoluble material (e.g., but not limited to, magnetic beads), and the second antibody may be a labeled antibody. The sandwich immunoassay procedure described above may be used in conjunction with the antibodies described below.

[0053] Turning now to specific non-limiting embodiments of this disclosure, embodiments of which include assays suitable for detecting individual biomarkers such as NT-proBNP or characteristic fragments thereof, and compositions / devices / kits containing the same, methods for generating and using the same, kits, and associated diagnostic tests. Certain non-limiting embodiments of this disclosure include preselective assays for detecting NT-proBNP in serum. Certain non-limiting embodiments of this disclosure advantageously reduce, eliminate, or substantially eliminate biotin interference in the reaction. In certain non-limiting embodiments, the preselective assay advantageously restricts the binding of biotin to the reagent being measured.

[0054] Certain non-limiting embodiments of this disclosure relate to methods for determining the presence, severity, and / or susceptibility to heart failure in an individual using a single biomarker. In certain non-limiting embodiments, this disclosure includes a method comprising the following process sequence: (a) forming a solid-phase immunoassay reagent by contacting one or more streptoacid-coated particles with one or more biotinylated antibodies to form a complex; and (b) separating the solid-phase immunoassay reagent from a lite immunoassay reagent and storing or packaging the solid-phase reagent separately from the lite reagent. In certain non-limiting embodiments, because the solid-phase reagent is present with the biotinylated antibodies in a pre-complexed form, there is essentially no opportunity for biotin interference from the biofluid sample. For NT-proBNP analytes, “no biotin interference” is defined as a percentage deviation of ≤10%. In certain non-limiting embodiments of this disclosure, the term “no biotin interference” includes no interference at biotin concentrations up to 3510 ng / mL.

[0055] This disclosure facilitates the diagnosis of HF, such as immediate or remote diagnosis, and assists healthcare providers in monitoring the status or progression of HF at two or more time points. Embodiments of this disclosure are applicable to outpatient settings, such as when a patient in need provides a biosample, or when one or more embodiments of this disclosure are used as part of an outpatient care strategy, such as when the medical service implementation of this disclosure does not require hospitalization, or where the patient is free to leave the healthcare facility once the service or procedure of this disclosure is completed. Non-limiting examples of outpatient care include home health services, annual physical examinations in physician offices or non-hospitalized clinic settings (e.g., medical offices, clinics, outpatient surgery centers, hospital outpatient departments, and other non-hospital centers). In some embodiments, embodiments of this disclosure are suitable for use by users in home outpatient care to promote patient independence, allow a transition from hospital care to home care, or as part of a home monitoring strategy such as cardiac monitoring. In embodiments, the assays of this disclosure can be applied to a single occurrence in an outpatient care setting, such as an outpatient facility where the patient does not need to stay for more than 1–24 hours, 24 hours, or 12 hours.

[0056] In this implementation, patients requiring this will provide biological samples, such as blood or serum, from an outpatient setting. The biological samples may be shared by an institution suitable for performing one or more assays or implementations of this disclosure to analyze the patient's health information, such as cardiac information. In this implementation, after sample collection, chemical reactions between the analytes and reagents in the patient's biological sample are performed under conditions suitable for performing the assays of this disclosure and resulting in the generation of various signals, or the signals measured by the analyzer are modulated.

[0057] This disclosure also provides kits comprising one or more anti-HF biomarker reagents and instructions for use (e.g., for therapeutic, preventative, or diagnostic purposes). In some non-limiting embodiments, the kit is used for in vitro diagnostic assays to diagnose HF. In some non-limiting embodiments, the one or more anti-HF biomarker reagents comprise antibody reagents. In some non-limiting embodiments, the one or more antibody reagents are labeled with a detectable portion. In some non-limiting embodiments, the kit further comprises a detection reagent (e.g., one or more acridinium ester molecules). In some non-limiting embodiments, the one or more antibody reagents are labeled with one or more acridinium ester molecules. In some non-limiting embodiments, the kit further comprises one or more secondary antibodies that specifically bind to one or more anti-HF biomarker antibody reagents. Additional kit embodiments are described in the examples below.

[0058] In some non-limiting embodiments, the instruction manual provides a suitable fit for ATELLICA. ® CI Analyzer (Siemens Healthineers USA, Malvern, PA), for example, the usage method of model 1900.

[0059] 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 standards.

[0060] In addition to the above, the kit may also include other components, such as (but not limited to) solvents or buffers, stabilizers or preservatives, and / or reagents for treating the conditions or symptoms described herein. Alternatively, other components may be included in the kit, but in a different composition or container than the anti-HF biomarker reagent. In such embodiments, the kit may include instructions for mixing the anti-HF biomarker reagent with other components, or for using the anti-HF biomarker together with other components. In some non-limiting embodiments, the provided instructions eliminate the time for any biotin in the sample to complex with the streptavidin-coated particles, and / or for the antibody interfering with biotin labeling to bind to the streptavidin-coated particles. In some non-limiting embodiments, the instructions include the use of streptavidin-coated particles characterized as pre-incubated with biotinylated antibodies, and in the form of a solid-phase reagent.

[0061] In some non-limiting embodiments, the kit used in accordance with this disclosure may include a reference or control sample, instructions for processing the sample, instructions for performing a test on the sample, and / or instructions for interpreting the results, as well as buffers and / or other reagents required for performing the test.

[0062] The methods and kits provided herein for detecting NT-proBNP in samples have sensitivity and specificity that make the test results sufficiently reliable and medically operable. The methods and kits described herein for detecting and / or diagnosing HF in subjects detect NT-proBNP with sensitivities 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 sensitivities of about 70%-100%, about 80%-100%, or about 90%-100%. In some non-limiting embodiments, the methods and kits provided herein can detect NT-proBNP with sensitivities and specificities of about 50%-100%, about 60%-100%, about 70%-100%, about 80%-100%, or about 90%-100%.

[0063] This document also provides compositions. In some non-limiting embodiments, the composition comprises one or more biotinylated antibodies bound to one or more streptavidin-coated solid supports (e.g., but not limited to, streptavidin-coated beads or particles). In some non-limiting embodiments, the assay comprises one or more streptavidin-coated particles, such as one or more bead substrates pre-incubated with one or more biotinylated antibodies to form one or more solid-phase reagents suitable for eliminating or substantially eliminating interference from free biotin in a biofluid sample. In some non-limiting embodiments, this disclosure adjusts the signal-to-noise ratio for NT-proBNP sandwich immunoassays suitable for high-throughput analyzers. In some non-limiting embodiments, because the solid-phase reagent is present with the biotinylated antibody in a pre-composite form, there is substantially no opportunity for interference from free or native biotin from the sample when the reagent is mixed with the sample. In some non-limiting embodiments, for example for NT-proBNP analytes, "no interference with biotin" is defined as a percentage deviation of ≤10%. In some non-limiting embodiments, this disclosure allows for the determination of NT-proBNP up to 3510 ng / mL of biotin without interference.

[0064] In some non-limiting embodiments, this immunoassay can be run on a high-throughput chemistry analyzer capable of performing more than 20, 30, 40, 50, 75, or 100 tests per hour. In some non-limiting embodiments, this immunoassay can be, for example, at ATELLICA… ®CI Analyzer (Siemens Healthineers USA, Malvern, PA), for example, the 1900 model.

[0065] In one form of diagnostic method, immunoassay, one or more specific binding species are used. A typical example is a sandwich immunoassay, in which two specific binding species (antibody or antigen) bind to non-overlapping epitopes of the analyte of interest. One of the specific binding species is typically attached 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 labeling allows for the detection of the analyte of interest by a variety of detection methods corresponding to the label used.

[0066] Another type of specific binding species covalently or frequently by adsorption binds to a solid or suspendable matrix (“solid phase”). Alternatively, it can 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 attached to the solid phase. This allows the specific binding species to bind to the solid via the interaction of the second binding pair (e.g., biotin-streptavidin).

[0067] The solid phase can be a macroscopic solid phase, such as (but not limited to) microburette orifices, tubes and balls in tubular devices, or a suspended solid phase, such as (but not limited to) beads, latex beads, magnetic latex beads, and other paramagnetic materials. Secondary binding types are typically labeled using tags. The interaction between the tag and the solid phase allows for the detection and quantification of the analyte of interest using a variety of detection methods corresponding to the tags employed.

[0068] Figure 1-2 The effects of existing sandwich immunoassays on analytes such as (but not limited to) NT-proBNP, along with biotin interference, have been confirmed. Figure 1As can be seen, the existing technique utilizes three reagents. Reagent 1 is a streptavidin-coated particle (e.g., but not limited to, streptavidin-coated magnetic beads); Reagent 2 is a biotinylated first antibody; and Reagent 3 is an acridinium-labeled second antibody. The two antibodies of Reagents 2 and 3 bind to non-overlapping epitopes of NT-proBNP, allowing each antibody to bind to a single molecule of NT-proBNP. When the three reagents are mixed with a biological sample suspected of containing NT-proBNP (e.g., but not limited to, plasma or serum), the two antibodies bind to NT-proBNP, and the biotinylated antibody binds to the streptavidin of the particle to form reaction product 1, wherein the acridinium ester is indirectly correlated with the solid-phase particle via the binding of the two antibodies to NT-proBNP. Therefore, reaction product 1 can be detected by a signal generated by the acridinium ester associated with the solid phase.

[0069] However, as Figure 2 As shown, when free biotin is present in a biological sample, it competes with biotinylated antibodies for binding to streptavidin-coated particles. This results in three reaction products: a detectable reaction product 1, a reaction product 2 containing NT-proBNP molecules bound by both antibodies (and therefore containing acridinium ester), and a reaction product 3 containing free biotin bound to streptavidin-coated particles. Therefore, a false low (or false negative) measurement is identified because a portion of the NT-proBNP present in the sample is not present in product 1 and is therefore undetectable, as the acridinium ester label bound to the two antibodies and the NT-proBNP analyte (product 2) cannot bind to the streptavidin-coated particles (product 3) for label detection.

[0070] In comparison, Figure 3 Non-limiting embodiments of kits, systems, and methods according to this disclosure for performing immunoassays of target analytes (e.g., but not limited to, NT-proBNP) are shown. In this embodiment, only two reagents are used: reagent 1 is a streptavidin-coated particle with a biotinylated first antibody pre-bound thereto, 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 with or substantially does not interfere with the formation of product 1, wherein the acridinium ester label is associated with the solid-phase particle by the NT-proBNP binding of the two antibodies.

[0071] In certain specific (but non-limiting) embodiments, the use of streptavidin-coated magnetic beads provides a rapid magnetic response time and is suitable for high-throughput and multiplexed assays. The streptavidin coating is contacted with one or more biotinylated antibodies under conditions suitable for binding to biotinylated antibodies. Therefore, the immunoassay of this disclosure can be a fully automated two-site sandwich immunoassay using direct chemiluminescence technology, employing constant or substantially constant amounts of two monoclonal antibodies. The solid phase contains a biotinylated monoclonal sheep anti-human antibody that binds specifically to NT-proBNP and is conjugated to streptavidin magnetic particles. The simplified 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 the patient sample and the amount of relative optical units (RLU) detected by the system.

[0072] 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 may be configured to output an HF biomarker score based on the received HF biomarker profile and / or the level of the NT-proBNP biomarker. In particular, the computer program may include instructions regarding the system's selection of appropriate subsequent steps, including additional medication, treatment, and / or additional testing for the subject.

[0073] In some non-limiting embodiments, the computer program may be configured such that the computer system can identify subjects for further testing (e.g., cardiac testing), subjects at risk of or with HF, and / or subjects receiving medication based on received data (e.g., NT-proBNP biomarker profile), and use the data to calculate an NT-proBNP biomarker score.

[0074] Figure 4 This is a block diagram of a computer system 1100 that can be used for the operations described above, according to a non-limiting embodiment. System 1100 includes a processor 1110, a memory 1120, a storage device 1130, and an input / output device 1140. Components 1110, 1120, 1130, and 1140 are interconnected using a system bus 1150. The system may optionally further include an analytical device 1160 for determining the levels of one or more biomarkers of this disclosure in a sample.

[0075] In some non-limiting embodiments, processor 1110 is capable of processing instructions for execution within system 1100. In one non-limiting embodiment, processor 1110 is a single-threaded processor. In another non-limiting embodiment, processor 1110 is a multi-threaded processor. Processor 1110 is capable of processing instructions stored in memory 1120 or storage device 1130, including receiving or sending information via input / output device 1140.

[0076] In some 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 cell. In another embodiment, memory 1120 is a non-volatile memory cell.

[0077] Storage device 1130 is capable of providing large-capacity storage for system 1100. In one non-limiting embodiment, storage device 1130 is a computer-readable medium.

[0078] Input / output device 1140 provides input / output operations to system 1100. In one non-limiting embodiment, input / output device 1140 includes a keyboard and / or pointing device. In one non-limiting embodiment, input / output device 1140 includes a display unit for displaying a graphical user interface.

[0079] System 1100 can be used to build a database. In some non-limiting embodiments, the methods of this disclosure are performed within system 1100, which is located within a chemical analyzer. For example, a computer program product may include instructions that cause processor 1110 to perform the steps of any method disclosed herein or otherwise contemplated.

[0080] Additionally, a non-transient computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including the methods provided herein. For example, a non-transient computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including any methods disclosed herein or otherwise contemplated. In some non-limiting embodiments, the non-transient computer-readable medium includes hard drives, external hard drives, optical discs, CDs, DVDs, and / or similar media for storing data. In some non-limiting embodiments, software disposed within a physical medium is suitable for the purposes of this document.

[0081] In some non-limiting embodiments, a non-transient computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including methods for determining the presence, severity, and / or susceptibility to heart failure (HF) in an individual, said methods comprising the steps of: incubating the assay component of this disclosure together with a biological sample, and determining the amount of NT-proBNP in the sample. Example

[0082] Examples are provided below. However, this disclosure should be understood as not being limited in its application to the specific experiments, results, and laboratory procedures disclosed below. Rather, the examples are provided merely as one of various implementations and are intended to be exemplary rather than exhaustive.

[0083] Example 1 In one non-limiting embodiment, this disclosure includes an NT-proBNP assay suitable for in vitro diagnostic use in the quantitative determination of N-terminal pro-brain natriuretic peptide (NT-proBNP) in human serum and plasma (EDTA and lithium heparin) using a chemical analyzer, such as (but not limited to) the ADVIA Centaur® XP system. In the emergency department (ED) and outpatient (OP) populations, NT-proBNP measurement is used as an adjunct in the diagnosis of heart failure (HF) in patients with clinically suspected new-onset or worsening HF, and for the evaluation of HF severity.

[0084] In one non-limiting embodiment, the materials present in the kit include: ●Simplified reagent (7.5 ml / pack) contains acridinium-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment antibody (~0.36 µg / ml) in buffer; bovine serum albumin (BSA); bovine gamma globulin; and preservatives.

[0085] ● Solid-phase reagent (20.0 ml / packet) containing a buffer containing biotin-labeled monoclonal sheep anti-human NT-proBNP antibody (~2 µg / ml) bound to streptavidin magnetic particles (~220 mg / L); BSA; bovine gamma globulin; sheep gamma globulin; and preservatives.

[0086] ●Optional Ancillary Well Reagent (7.5 ml / pack) contains buffer, BSA, bovine gamma globulin, sheep gamma globulin, and preservatives.

[0087] In some non-limiting embodiments, the assay of this disclosure is a fully automated two-site sandwich immunoassay using direct chemiluminescence technology, employing constant amounts of two monoclonal antibodies. The solid phase contains a biotinylated monoclonal sheep anti-human antibody that specifically binds to NT-proBNP and is conjugated to streptavidin magnetic particles. The simplified reagent contains an acrid ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment that is specific to NT-proBNP. There is a direct relationship between the amount of NT-proBNP present in the patient sample and the amount of relative optical units (RLU) detected by the system.

[0088] Non-restrictive procedures for collecting samples or specimens may involve one or more of the following optional steps: following general precautions when collecting samples; handling all samples as if they could transmit disease; following recommended procedures for collecting diagnostic blood samples by venipuncture; following the instructions for use and processing provided with your sample collection device; allowing the blood sample to coagulate completely before centrifugation; and / or always keeping the tube cap on.

[0089] Non-restrictive procedures for storing samples or specimens may involve one or more of the following optional steps: serum samples stored on clots are stable at 2–8°C for up to 24 hours after centrifugation; isolated samples are stable at room temperature for up to 3 days and at 2–8°C for up to 4 days; isolated samples are stable at ≤ -20°C for up to 12 months; avoid more than one freeze-thaw cycle; do not store in a frost-free freezer; and / or thoroughly mix the thawed sample and centrifuge it before use.

[0090] Non-restrictive procedures for transporting samples or specimens may involve one or more of the following optional steps: packaging and labeling the samples for transport in accordance with applicable federal and international regulations covering the transport of clinical samples and pathogens; and / or transporting frozen samples if the samples may be subjected to temperatures >25°C during transport.

[0091] The non-limiting procedure for sample preparation may involve one or more of the following optional steps: the determination requires 20 µL of sample for a single determination. This volume does not include volumes that are unavailable in the sample container or additional volumes required when performing repeat tests or other tests on the same sample; the sample volume required for onboard dilution differs from the sample volume required for a single determination of an undiluted sample; samples with significant contamination are not used; before placing the sample on the system, ensure that the sample is free of the following: air bubbles or foam, fibrin or other particulate matter; and / or remove particulate matter by centrifugation as recommended by CLSI guidelines and the collection device manufacturer.

[0092] Non-limiting assay procedures may involve one or more of the following optional steps. The system (including a high-throughput chemical analyzer) automatically performs the following steps: dispensing 20 µL of sample into a cuvette; dispensing 200 µL of solid phase and 75 µL of auxiliary well reagent, followed by incubation at 37°C for 3 min; dispensing 75 µL of simplified reagent, followed by incubation at 37°C for 6 min; performing a washing sequence using a washing reagent (e.g., but not limited to ADVIA Centaur Wash 1); dispensing 300 µL each of acid and base reagents (e.g., but not limited to ADVIA Centaur Acid Reagent and ADVIA Centaur Base Reagent) to initiate the chemiluminescent reaction; and / or reporting the results.

[0093] Non-limiting statements regarding the preparation of reagents for use in automated systems include: all reagents are liquid and ready-to-use. Reagents need to be mixed before loading the package onto the system. For information on mixing reagents, refer to the system's online help.

[0094] Non-limiting instructions for preparing an automated system to perform the determinations of this disclosure include: ensuring sufficient material is loaded onto the system; guidance on required reagents, referring to the provided materials and required but not provided materials.

[0095] Non-restrictive instructions for setting the master curve definition include: before initiating calibration for each new batch of reagents, input the master curve values ​​by scanning the master curve card.

[0096] Non-limiting instructions for performing calibration include: for calibration of a specific non-limiting implementation of the assay, use the calibrator provided with each kit. The calibrator provided in the assay kit may only be used with the reagent batch provided in the same kit.

[0097] Non-limiting instructions for calibration frequency include: perform calibration if one or more of the following conditions are met: at the end of a 36-day calibration interval; when the lot number of the primary reagent package is changed; when indicated by quality control results; and after major maintenance or repair, if indicated by quality control results. Follow government regulations or certification requirements regarding calibration frequency. Individual laboratory quality control procedures and protocols may require more frequent calibrations.

[0098] Non-limiting instructions for preparing the calibrator include one or more of the following optional steps: Add 2.0 mL of reagent water to each vial. Replace the cap. Allow the vials to stand at room temperature for 30 minutes to allow the lyophilized material to dissolve. Gently mix the vials and invert them to ensure homogeneity of the material. For extended storage, aliquot and seal tightly. reagentsThe specified stability limits apply to the storage of reconstructed materials. Do not store in a frost-free freezer. Allow the materials to thaw completely before using the frozen calibrator. Gently mix the vials and invert them to ensure material homogeneity. reagents Use the calibrator within the stability limits specified in the specification, and discard any remaining material.

[0099] Non-limiting instructions for performing the calibration procedure include: ensuring that the appropriate master curve and calibrator allocation values ​​are entered into the system. Refer to the system's online help for information on defining the master curve and entering calibrator values. Load the reagents required for the assay. Arrange the calibrators. Label the two sample containers with barcodes: one for the low-calibrator and one for the high-calibrator. Place the barcode labels on the sample containers with the readable characters vertically oriented. Barcode labeling is batch-specific. Do not use barcode labels from one batch of calibrators for any other batch. Gently mix the products and dispense sufficient volume of each calibrator into the appropriate sample container. Avoid air bubbles. The required sample volume 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. Dispose of any remaining calibrator in the sample containers after 6 hours. Do not refill or reuse sample containers. Do not put any calibrator material back into the original containers.

[0100] Non-limiting instructions for performing quality control include: For quality control of certain non-limiting embodiments of the assays of this disclosure, appropriate quality control material with a minimum of two levels (low and high) of known analyte concentration shall be used at least once per day during the analysis of the sample. Use the quality control material according to the quality control instructions. Additional quality control material may be used at the laboratory's discretion. Additionally, quality control shall be performed: after effective calibration; when using a new batch of reagents; and when troubleshooting test results that do not match clinical conditions or symptoms. Follow government regulations or certification requirements regarding the frequency of quality control. Individual laboratory quality control procedures and protocols may require more frequent quality control testing. Acceptable performance is achieved when the obtained analyte values ​​are within the system's expected control intervals, as indicated by the manufacturer of the control material or within intervals determined by the internal laboratory quality control procedures. If the obtained results do not fall within acceptable limits, follow your laboratory's quality control procedures. For information on entering quality control definitions, refer to the system's online help.

[0101] Non-restrictive instructions for taking corrective action include: if the quality control results do not fall within the expected control interval, do not report the results. Perform corrective action according to the established laboratory protocol. Refer to the system's online help for proposed protocols.

[0102] result Result Calculation The system uses the calculation procedures described in the system's online help to determine the results. The system reports results in pg / mL (common units) or pmol / L (SI units), depending on the units defined at the time of the assay. Conversion formula: 1 pg / mL = 0.118 pmol / L. For information on results outside the specified measurement interval, refer to [link to relevant documentation]. Measurement interval .

[0103] dilution Measurement intervals are 35–35,000 pg / mL (4.13–4130 pmol / L). Refer to the system's online help for information on dilution options. Dilute and retest samples with NT-proBNP levels >35,000 pg / mL (4130 pmol / L) to obtain accurate results. For automated dilution, do the following: Load a multi-diluent (e.g., but not limited to, ADVIACentaur Multi-Diluent 1). Ensure sufficient sample volume is available. Refer to the table below. Select the appropriate dilution factor. For automated dilution, enter a dilution point ≤ 35,000 pg / mL (4130 pmol / L).

[0104] Table 1 sample Dilution Sample volume (µL) serum and plasma 1:5 40 serum and plasma 1:10 30

[0105] If patient results exceed the measurement interval when using auto-dilution, or if the laboratory protocol requires manual dilution, then manually dilute the patient sample. For manual dilution, do the following: prepare the manual dilution using a multi-diluent (e.g., but not limited to ADVIA Centaur Multi-Diluent 1 vial). In the implementation plan, refer to the operating manual and the discussion related to optional materials. For information on ordering tests for manually diluted samples, refer to the system's online help. Ensure that the results are mathematically corrected for dilution. If a dilution factor was entered when scheduling the test, the system automatically calculates the results.

[0106] Interpretation of Results Results from the assays disclosed herein should be interpreted in conjunction with appropriate clinical guidelines and in conjunction with the patient's medical history, clinical presentation, and other findings. Guidelines recommend the use of natriuretic peptides for the diagnosis or exclusion of heart failure in both emergency department (ED) and outpatient (OP) settings. The performance of the assays disclosed herein is assessed separately in these settings using the disclosed age-independent and age-dependent cutoff values.

[0107] Emergency Department (ED) population For patients presenting in an ED environment due to clinical suspicion of HF, NT-proBNP test results should be interpreted as indicated in Table 2.

[0108] Table 2

[0109] Elevated or decreased natriuretic peptide (NT-proBNP) levels can be caused by conditions that can confuse 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 insufficiency, stroke, and sepsis can elevate NT-proBNP levels in the absence of acute heart failure. Additionally, obesity, acute pulmonary edema, pericarditis, and cardiac tamponade are associated with decreased NT-proBNP levels.

[0110] outpatient group In an outpatient setting, natriuretic peptide testing is best used to exclude the diagnosis of HF. Therefore, a low exclusion cutoff value is required, which increases sensitivity and negative predictive value, as these patients may present with limited, less severe HF symptoms. For non-bedridden patients presenting to an outpatient facility with clinically suspected HF (previously undiagnosed), test results should be interpreted as indicated in Table 3.

[0111] Table 3

[0112] Clinical conditions such as acute coronary syndrome, atrial fibrillation, pulmonary embolism, valvular heart disease, myocarditis, pulmonary hypertension, renal insufficiency, stroke, and sepsis can raise NT-proBNP levels in the absence of heart failure.

[0113] limitation Patient samples may contain heterophilic antibodies, which can react in immunoassays and cause false elevations or decreases in results. This assay is designed to minimize interference from heterophilic antibodies. Additional information may be needed for diagnosis.

[0114] Expected values ​​in healthy individuals (Table 4) Predicted values ​​were determined nonparametrically in a population of 723 surface-healthy subjects (362 women and 361 men) without HF, using the ADVIA Centaur® XP system, in accordance with CLSI document EP28-A3c.

[0115] Table 4

[0116] As with all in vitro diagnostic assays, each laboratory should establish its own reference ranges for the diagnostic assessment of patient outcomes. These values ​​should be considered as guidelines only.

[0117] Disease Research Group ED cohort: A total of 3128 participants who presented to the ED cohort with signs and symptoms of HF were included. Of these, 1148 participants (476 women and 672 men) were diagnosed with acute HF and included in the disease study cohort. NT-proBNP values ​​based on age group (Table 5) and NYHA functional classification (Table 6) are summarized by subgroup for participants with acute HF in Tables 5-6. Men with acute HF are summarized in Table 7, while women with acute HF are summarized in Table 8.

[0118] Table 5: ED group - all subjects with acute HF

[0119] Table 6

[0120] Table 7 a The cutoff value is not applicable to the indicated age group.

[0121] Table 8

[0122] ED population – All subjects with acute HF (e.g., NYHA functional classification). NYHA classifications for the ED population with acute HF are provided in Table 9. Men with acute HF (NYHA functional classification) are shown in Table 10, while women with acute HF are shown in Tables 11-12.

[0123] Table 9 a Unavailable: Insufficient sample size for calculating percentiles. 12 b The NYHA classification could not be determined for 16 subjects. c A male subject identified as an outlier with an abnormally high PBNPII value was excluded from the NYHA Class I classification generalization.

[0124] Table 10 aThe NYHA classification could not be determined for 10 subjects.

[0125] Table 11

[0126] Table 12 a Unavailable: Insufficient sample size for calculating percentiles. 12 Outpatient (OP) group A total of 1033 participants who presented to the OP setting with signs and symptoms of HF were enrolled. Of these, 185 participants (102 women and 83 men) were diagnosed with new-onset HF and included in the disease study group. NT-proBNP values ​​were summarized for participants with new-onset HF based on age groups (Tables 13–15) and NYHA functional classifications (Tables 16–18). Table 13 summarizes the OP population – all participants with new-onset HF. Men with new-onset HF are summarized in Table 14, while women with new-onset HF are summarized in Table 15.

[0127] The NYHA classification for the OP population with new-onset HF is provided in Table 16. Men with new-onset HF (NYHA functional classification) are shown in Table 17, while women with new-onset HF are shown in Table 18.

[0128] Table 13

[0129] Table 14

[0130] Table 15

[0131] Table 16 a Unavailable: Insufficient sample size for calculating percentiles. 12 Table 17 a Unavailable: Insufficient sample size for calculating percentiles. 12 Table 18 a Unavailable: Insufficient sample size for calculating percentiles. 12 OP group – receiver operation characteristic curve The recipient operating characteristic (ROC) curves shown in Table 5 present the clinical sensitivity and specificity for the 185 enrolled patients diagnosed with new-onset HF and the 848 subjects without HF. The area under the ROC curve for NT-proBNP measurement was 0.839, with a 95% confidence interval of 0.804 to 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 quantitation (LoQ). Results below the measurement interval are reported as <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 quantitation (LoQ): 35 pg / ml (4.13 pmol / L) LoB and LoD values ​​are representative data. LoB corresponds to the highest measurement result most likely to be observed with a 95% probability for a blank sample. LoD corresponds to the lowest concentration of NT-proBNP detectable with a 95% probability. LoQ corresponds to the lowest amount of NT-proBNP in the sample, below which the in-laboratory CV is 20%. Detection capability is determined according to CLSI document EP17-A2.

[0134] Clinical manifestations A total of 3,128 participants presenting with signs and symptoms of acute heart failure (HF) at ED were prospectively enrolled in a multicenter clinical evaluation of a non-limiting implementation of the NT-proBNP assay disclosed herein. The diagnosis and severity of HF were determined by an independent central adjudication panel of expert clinicians (cardiologists). 1,148 participants were adjudicated as having acute HF, and 1,980 participants were adjudicated as not having HF.

[0135] At the time of inclusion, descriptive statistics regarding NT-proBNP test results (pg / ml) were determined for the ED population.

[0136] Table 19 summarizes the baseline characteristics of all ED groups, while Table 20 summarizes the baseline characteristics of ED groups by age group.

[0137] Table 19 a 25th percentile. b 75th percentile.

[0138] Table 20 a 25th percentile. b 75th percentile.

[0139] Likelihood ratio NT-proBNP results were stratified into three categories based on an overall exclusion cutoff of 300 pg / ml and an age-specific inclusion cutoff, and compared with the definitive diagnosis of acute heart failure (HF). The pre-test risk (or prevalence) of HF in this study was 36.7%. The risk of HF increased to 67.2% (post-test risk) for subjects with a positive NT-proBNP result relative to this pre-test risk. Likelihood ratios compared the probability of an NT-proBNP test result given for acute heart failure to the probability of an NT-proBNP test result given for heart failure. For a positive NT-proBNP test result, a likelihood ratio of 3.53 indicated that individuals with acute HF were 3.5 times more likely to have a positive test result than those without acute HF. The post-test risk of HF decreased to 24.3% for subjects with indeterminate results, and the probability in the HF population was nearly half that in the negative diagnosis population (LR 0.55). For a negative NT-proBNP test result, a likelihood ratio of 0.07 indicates that an individual with acute HF is 0.07 times more likely (less than 1 / 10) to have a negative test result than an individual without acute HF.

[0140] The analysis of clinical manifestations between the non-limiting embodiments of the NT-proBNP assay and the definitive diagnosis in this disclosure was performed using a 2×3 contingency table format. Tables 21-32 summarize the NT-proBNP results relative to the definitive diagnosis (positive or negative) regarding acute HF using the following criteria: The inclusion cutoff value was greater than the age-specific threshold (positive). Between inclusion and exclusion cutoff values ​​(uncertain) Below the exclusion cutoff value (negative) LR should only be used for the ED population.

[0141] Clinical concordance between NT-proBNP results and the diagnosis of acute HF in the non-limiting embodiments of the assays disclosed herein. Table 21 summarizes the results for all sexes and age groups pooled together; Table 22 summarizes the results by age group; Table 23 summarizes the results for male subjects by age group; and Table 24 summarizes the results for female subjects by age group. Analyses were also performed for relevant clinical subgroups of the ED population, as shown in Tables 25-32.

[0142] Table 21 a Age-specific cutoff values ​​were included. b 95% confidence interval.

[0143] Example The analysis was performed by merging all age groups and then by subgrouping based on age group and sex.

[0144] Table 22 a 95% confidence interval.

[0145] Table 23 a 95% confidence interval.

[0146] Table 24 a 95% confidence interval.

[0147] Table 25 ED group - those with BMI < 30 kg / m 2 Clinical consistency between NT-proBNP results and the diagnosis of acute HF in patients a 95% confidence interval. b not applicable.

[0148] Table 26 ED group - those with BMI ≤30 kg / m 2 Clinical consistency between NT-proBNP results and the diagnosis of acute HF in patients a 95% confidence interval.

[0149] Table 27 ED population - those with eGFR < 60 mL / min / 1.73 m 2 Clinical consistency between NT-proBNP results and the diagnosis of acute HF in patients a 95% confidence interval. b not applicable.

[0150] Table 28 ED population - in individuals with eGFR ≥ 60 mL / min / 1.73 m 2 Clinical consistency between NT-proBNP results and the diagnosis of acute HF in patients a 95% confidence interval.

[0151] Table 29 ED population – Clinical consistency between NT-proBNP results and the definitive diagnosis of acute heart failure in patients with a history of heart failure. a 95% confidence interval.

[0152] Table 30 ED population – Clinical consistency between NT-proBNP results and the definitive diagnosis of acute heart failure in patients without a history of heart failure. a 95% confidence interval.

[0153] Table 31 ED population – Clinical consistency between NT-proBNP results and the adjudicated diagnosis of acute heart failure in patients with comorbidities. a 95% confidence interval. b Subjects included those with at least one of the following comorbidities: diabetes mellitus, renal insufficiency (eGFR <60 mL / min / 1.73 m³ / min). 2 ) and / or high blood pressure.

[0154] Table 32 Clinical consistency between NT-proBNP results and the definitive diagnosis of acute heart failure in the ED population in patients without comorbidities. a 95% confidence interval. b Subjects included those with at least one of the following comorbidities: diabetes mellitus, renal insufficiency (eGFR <60 mL / min / 1.73 m³ / min). 2 ) and / or high blood pressure. c not applicable.

[0155] The following statements are based on performance in clinical studies: With a BMI ≥ 30 kg / m 2 Patients with a BMI < 30 kg / m² 2 The false negative rate was higher among patients with a BMI ≥ 30 kg / m². Of those false negatives (44 / 1148), 41 (93%) came from patients with a BMI ≥ 30 kg / m². 2 Of the patients, 1 (2.2%) came from those with a BMI <30 kg / m². 2 Of the patients, 2 (4.4%) were from patients with unknown BMI.

[0156] With eGFR < 60 mL / min / 1.73 m 2 Patients with eGFR ≥ 60 mL / min / 1.73 m 2 The false positive rate was higher among patients diagnosed as not having acute HF with eGFR <60 mL / min / 1.73 m 2 Of the total 492 patients, 226 (45.9%) had NT-proBNP concentrations ≥ ASC. Among those diagnosed as not having acute HF, those with eGFR ≥ 60 mL / min / 1.73 m 2 Of the total 1427 patients, 251 (17.6%) had NT-proBNP concentrations ≥ ASC. Sixty-one (61) patients (3.1%) who were ruled not to have acute HF had unknown eGFR.

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

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

[0159] Clinical manifestations in the OP population A total of 1033 patients with signs and symptoms of new-onset HF were prospectively enrolled in a multicenter clinical evaluation of a non-limiting implementation of the NT-proBNP assay disclosed herein. Clinical performance was evaluated using a single cutoff value of 125 pg / ml.

[0160] The diagnosis and severity of HF were determined by an independent central adjudication panel of expert clinicians (cardiologists). One hundred and eighty-five (185) subjects were adjudicated as having new-onset HF, and 848 subjects were adjudicated as not having HF.

[0161] Table 33 OP population - baseline characteristics a 25th percentile. b 75th percentile.

[0162] Clinical sensitivity and specificity Clinical sensitivity and specificity for the OP population and relevant clinical subgroups were determined by comparing the performance of a non-limiting embodiment of the NT-proBNP assay of this disclosure with the definitive diagnosis. Using a single cutoff of 126 pg / mL, the analysis consisted of calculations of clinical sensitivity, clinical specificity, positive predictive value (PPV), and negative predictive value (NPV) relative to the definitive diagnosis of new-onset HF. Results are summarized in Tables 34-38.

[0163] Clinical sensitivity and specificity were determined in accordance with CLSI document EP12-A2.

[0164] Table 34 Clinical manifestations of the OP group - all subjects a 95% confidence interval.

[0165] Table 35 Clinical manifestations of OP population by gender and age group a 95% confidence interval.

[0166] Table 36 Clinical manifestations of the OP group based on BMI a BMI status unavailable (N = 6) b 95% confidence interval.

[0167] Table 37 Clinical manifestations of the OP population based on eGFR a eGFR status is unavailable (N = 627) b 95% confidence interval.

[0168] Table 38 Clinical manifestations of comorbid opioids a Subjects with at least one of the following comorbidities were included: diabetes mellitus, renal insufficiency (eGFR < 60), and / or hypertension. Comorbidity status was not available (N = 17). b 95% confidence interval.

[0169] Accuracy Accuracy was determined according to CLSI document EP05-A3. Samples were run twice daily in duplicate for 20 days. The results in Table 39 are representative of the performance of the assay.

[0170] Table 39 a Number of measurements. b Standard deviation. c Correlation coefficient.

[0171] The measurement is specified to have the following accuracy.

[0172] Reproducibility Reproducibility was determined according to CLSI document EP05-A3. Samples (N = 90) were run in triplicate at three locations twice daily for five days. The results shown in Table 40 are representative of the performance of the assay.

[0173] Table 40 a Standard deviation. b Correlation coefficient.

[0174] Sample equivalence Sample equivalence was determined using the Passing-Bablok regression model according to CLSI document EP09c-ed3. Consistency of sample types can vary depending on the study design and the test population.

[0175] Table 41 a The number of samples tested. b Correlation coefficient.

[0176] The assay was designed with a correlation coefficient of 0.950–1.000, a slope of 1.00 ± 0.05, and an intercept of ≤ 6 pg / mL (0.708 pmol / L).

[0177] interference Hemolysis, jaundice, hyperlipidemia (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. At NT-proBNP concentrations of 123–137 pg / mL (14.5–16.2 ​​pmol / L) and 1612–1813 pg / mL (190–214 pmol / L), the deviation for these substances is less than 10%.

[0178] Table 42

[0179] 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. At NT-proBNP concentrations of 116–163 pg / mL (13.7–19.2 pmol / L) and 1478–2225 pg / mL (174–263 pmol / L), the deviations due to these substances do not exceed 10%.

[0180] Table 43

[0181] Cross-reactivity Cross-reactivity was determined according to CLSI document EP07-ed3. The cross-reactive compounds 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).

[0182] Table 44

[0183] linear Linearity tests were performed according to CLSI document EP06-ed2. Determinations were linear for measurement intervals of 35–35,000 pg / mL (4.13–4130 pmol / L).

[0184] Airborne dilution recovery rate Serum samples were diluted on-board using Multi-Diluent 1 on the ADVIA Centaur XP System. The results in Table 45 are representative of the assay performance.

[0185] Table 45

[0186] High-dose hook effect High NT-proBNP concentrations can lead to an anomalous reduction in RLU (high-dose hook effect). In this assay, patient samples with NT-proBNP concentrations above the measurement interval and up to 300,000 pg / mL (35,400 pmol / L) will be reported as >35,000 pg / mL (4,130 pmol / L).

[0187] standardization The determination is traceable to an internal standard manufactured using high-purity materials. The allocation values ​​for the calibrators are also traceable to this standardization. Currently, no reference standard is available for this determination.

[0188] Example 2 Tables 46-47 summarize the characteristics and reagents of one non-limiting embodiment of the kit constructed according to this disclosure. The non-assay-related reagents listed (i.e., washing reagents, multi-dilution agents, etc.) are available, for example but not as a limitation, from Siemens Healthineers (Malvern, PA).

[0189] Table 46

[0190] Table 47

[0191] It should be noted that in some non-limiting embodiments, the simplified reagent, solid-phase reagent, auxiliary well reagent, calibrator, multiple diluent, and / or washing reagent can be stored unopened at 2-8°C. Additionally, in some non-limiting embodiments, the simplified reagent, solid-phase reagent, and / or auxiliary well reagent are substantially stable for at least 36 days after onboard operation.

[0192] Example 3 This embodiment includes a comparison of observed biotin interference between a non-limiting embodiment of the prior art three-reagent NT-proBNP assay and the present disclosure's two-reagent NT-proBNP assay. As can be seen, in the prior art three-reagent NT-proBNP assay, -28% to -23% biotin interference was observed at a biotin concentration of 200 ng / mL. In contrast, even at a biotin concentration of 17x, below which significant biotin interference was observed in the prior art assay, virtually no biotin interference (defined as a percentage deviation of ≤10%) was observed in the present disclosure's assay.

[0193] Table 48 Measurement Biotin test concentration Control pg / mL Test pg / mL %deviation Existing three-reagent determination 75 ng / mL 123 116 -6% Existing three-reagent determination 75 ng / mL 1783 1751 -2% Existing three-reagent determination 200 ng / mL 138 99 -28% Existing three-reagent determination 200 ng / mL 2553 1978 -23% New two-reagent assay 3510 ng / mL 121 127 5.0% New two-reagent assay 3510 ng / mL 1709 1651 -3.4%

[0194] Example 4 This embodiment includes a comparison of observed biotin interference between a non-limiting embodiment of the prior art three-reagent NT-proBNP assay and the present disclosure two-reagent NT-proBNP assay. As can be seen, the acceptable percentage deviation observed for the concentration in this assay occurs at a biotin concentration at least about 50x that is the level at which an acceptable percentage deviation is observed in the prior art assay.

[0195] Table 49 Measurement Biotin test concentration Control pg / mL Test pg / mL %deviation Existing three-reagent determination 75 ng / mL 123 116 -6% Existing three-reagent determination 75 ng / mL 1783 1751 -2% New two-reagent assay 3510 ng / mL 121 127 5.0% New two-reagent assay 3510 ng / mL 1709 1651 -3.4%

[0196] Non-restrictive illustrative implementation plan Illustrative Embodiment 1. A kit for determining the amount of NT-proBNP in a biological sample, the kit comprising: (a) a solid-phase reagent containing a solid support having a first anti-NT-proBNP antibody or a binding fragment thereof directly or indirectly attached thereto; and (b) a simplified reagent containing a second anti-NT-proBNP antibody or a binding fragment thereof labeled with acridinium ester; and 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.

[0197] Illustrative Embodiment 2. The kit of Illustrative Embodiment 1, wherein the first anti-NT-proBNP antibody or its binding fragment is biotinylated, and wherein at least a portion of the surface of the solid support is coated with a biotin-specific binding molecule, thereby indirectly attaching the first anti-NT-proBNP antibody or its binding fragment to the solid support.

[0198] Illustrative Embodiment 3. The kit of Illustrative Embodiment 2, wherein the biotin-specific binding molecule is selected from streptoavidin, traptavidin and avidin.

[0199] Illustrative Embodiment 4. The kit of Illustrative Embodiment 3, wherein the biotin-specific binding molecule is streptavidin.

[0200] Illustrative Embodiment 5. A kit of any one of Illustrative Embodiments 1-4, wherein the solid support comprises magnetic particles and / or latex particles.

[0201] Illustrative Embodiment 6. A kit of any one of Illustrative Embodiments 1-5, wherein the solid-phase reagent of (a) comprises a biotinylated monoclonal sheep anti-human antibody specific for NT-proBNP conjugated to magnetic particles, said magnetic particles having streptavidin coated on at least a portion of their surface.

[0202] Illustrative Implementation Scheme 7. A kit of any one of Illustrative Implementation Schemes 1-6, wherein the simplified reagent of (b) contains an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP.

[0203] Illustrative Implementation Scheme 8. A kit of any one of Illustrative Implementation Schemes 1-7, wherein (a) and (b) are packaged separately.

[0204] Illustrative Implementation 8A. A kit for any one of Illustrative Implementations 1-8, wherein the kit is configured for an emergency department environment.

[0205] Illustrative Implementation Scheme 8B. A kit for any one of Illustrative Implementation Schemes 1-8A, wherein the kit is configured for an outpatient setting.

[0206] Illustrative Implementation 8C. A kit for any one of Illustrative Implementations 1-8B, wherein the kit is configured for emergency department and / or outpatient settings.

[0207] Illustrative Embodiment 9. A method for determining the presence, severity, and / or susceptibility to heart failure (HF) in an individual, the method comprising the steps of: (a) simultaneously or in whole or in part sequentially combining the following to form a reaction mixture: (i) a biofluid sample; (ii) a solid-phase reagent comprising a solid support having a first anti-NT-proBNP antibody or a binding fragment thereof attached thereto; and (iii) a simplified reagent comprising a second anti-NT-proBNP antibody or a binding fragment thereof labeled with acridinium ester, wherein the first and second anti-NT-proBNP antibodies or binding fragments thereof bind to non-overlapping epitopes of NT-proBNP; (b) incubating the reaction mixture under conditions that allow (ii) and (iii) to bind with NT-proBNP present in the biofluid sample to form immune complexes; and (c) measuring the amount of the formed immune complexes to obtain a measurement of NT-proBNP in the sample.

[0208] Illustrative Implementation Scheme 10. The method of Illustrative Implementation Scheme 9 further includes the following steps: (d) obtaining an HF score using a mathematical algorithm based on the measured value of NT-proBNP in the sample.

[0209] Illustrative Embodiment 11. The method of Illustrative Embodiment 9 or 10, wherein the biological fluid sample is selected from blood, serum, plasma, and combinations thereof.

[0210] Illustrative Embodiment 12. A method of any one of Illustrative Embodiments 9-11, wherein the first anti-NT-proBNP antibody or its binding fragment is biotinylated, and wherein at least a portion of the surface of the solid support is coated with a biotin-specific binding molecule, thereby indirectly attaching the first anti-NT-proBNP antibody or its binding fragment to the solid support.

[0211] Illustrative Embodiment 13. The method of Illustrative Embodiment 12, wherein the biotin-specific binding molecule is selected from streptoavidin, traptavidin, and avidin.

[0212] Illustrative embodiment 13A. The method of illustrative embodiment 13, wherein the biotin-specific binding molecule is streptavidin.

[0213] Illustrative Embodiment 14. The method of any one of Illustrative Embodiments 9-13A, wherein the solid support comprises magnetic particles and / or latex particles.

[0214] Illustrative Embodiment 15. A method of any one of Illustrative Embodiments 9-14, wherein the solid-phase reagent of (ii) comprises a biotinylated monoclonal sheep anti-human antibody specific for NT-proBNP conjugated to magnetic particles, said magnetic particles having streptavidin coated on at least a portion of their surface.

[0215] Illustrative Embodiment 16. The method of any one of Illustrative Embodiments 9-15, wherein the simplified reagent of (iii) comprises an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific to NT-proBNP.

[0216] Illustrative Implementation Scheme 17. The method of any one of Illustrative Implementation Schemes 9-16, wherein substantially no interference from biotin present in the biological fluid sample is observed.

[0217] Illustrative Implementation Scheme 17A. The method of Illustrative Implementation Scheme 17, wherein there is essentially no biotin interference defined as a percentage deviation of ≤ 10%.

[0218] Illustrative Embodiment 17B. The method of Illustrative Embodiment 17 or 17A, wherein at biotin concentrations up to about 3510 ng / ml in a biological fluid sample, substantially no biotin interference is observed.

[0219] Illustrative Implementation Scheme 18. The method of any one of Illustrative Implementation Schemes 9-17, wherein at least one of steps (b), (c) and (d) is performed on an automated analyzer.

[0220] Illustrative Embodiment 19. The method of Illustrative Embodiment 18, wherein step (c) is further defined as measuring the amount of relative optical units (RLU), wherein the amount of RLU is proportional to the amount of NT-proBNP in the sample.

[0221] Illustrative Implementation 19A. A method of any one of Illustrative Implementations 9-19, wherein the method is performed in an emergency department setting.

[0222] Illustrative Embodiment 19B. A method of any one of Illustrative Embodiments 9-19A, wherein said method or a portion thereof is performed in an outpatient setting. For example, samples may be obtained in an outpatient setting or from outpatient strata and shared with institutions suitable for performing the methods of this disclosure or using the kits of this disclosure. In this embodiment, sharing may be accomplished by sending samples to institutions under conditions suitable for transporting biological materials.

[0223] Illustrative Implementation Scheme 19C. The method of any one of Illustrative Implementation Schemes 9-19B, wherein the method is performed in an emergency department, point-of-care and / or outpatient setting.

[0224] Illustrative Embodiment 20. A non-transient computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including any one of the methods in illustrative embodiments 9-19C.

[0225] Illustrative Embodiment 21. A kit for determining the amount of NT-proBNP in a biological sample, the kit comprising: (a) a solid-phase reagent containing a biotinylated monoclonal sheep anti-human antibody specific for NT-proBNP conjugated to magnetic particles, said magnetic particles having streptavidin coated on at least a portion of their surface; (b) a simplified reagent containing an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific for NT-proBNP; and wherein the antibody of (a) and the F(ab')2 fragment of (b) bind to non-overlapping epitopes of NT-proBNP, thereby forming an immune complex comprising (a), (b) and NT-proBNP.

[0226] Illustrative Implementation Scheme 22. The kit of Illustrative Implementation Scheme 21, wherein (a) and (b) are packaged separately.

[0227] Illustrative embodiment 22A. A kit for illustrative embodiment 21 or 22, wherein the kit is configured for an emergency department environment.

[0228] Illustrative Implementation Scheme 22B. A kit for any one of Illustrative Implementation Schemes 21-22A, wherein the kit is configured for an outpatient setting.

[0229] Illustrative Embodiment 23. A method for determining the presence, severity, and / or susceptibility to heart failure (HF) in an individual, the method comprising the steps of: (a) simultaneously or in whole or in part sequentially combining the following to form a reaction mixture: (i) a biological fluid sample; (ii) a solid-phase reagent comprising a biotinylated monoclonal sheep anti-human antibody specific for NT-proBNP conjugated to magnetic particles having streptavidin coated on at least a portion of its surface; (iii) a simplified reagent comprising an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific for NT-proBNP, wherein the antibody of (ii) and the F(ab')2 fragment of (iii) bind to non-overlapping epitopes of NT-proBNP; and (b) incubating the reaction mixture under conditions allowing (ii) and (iii) to bind with 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.

[0230] Illustrative Implementation Scheme 24. The method of Illustrative Implementation Scheme 23 further includes the following steps: (d) obtaining an HF score using a mathematical algorithm based on the measured value of NT-proBNP in the sample.

[0231] Illustrative Embodiment 25. The method of Illustrative Embodiment 23 or 24, wherein the biological fluid sample is selected from blood, serum, plasma and combinations thereof.

[0232] Illustrative Implementation Scheme 26. The method of any one of Illustrative Implementation Schemes 23-25, wherein substantially no interference from biotin present in the biological fluid sample is observed.

[0233] Illustrative Implementation Scheme 26A. The method of Illustrative Implementation Scheme 26, wherein there is essentially no biotin interference defined as a percentage deviation of ≤ 10%.

[0234] Illustrative Embodiment 26B. The method of Illustrative Embodiment 26 or 26A, wherein at biotin concentrations up to about 3510 ng / ml in a biological fluid sample, substantially no biotin interference is observed.

[0235] Illustrative Implementation Scheme 27. The method of any one of Illustrative Implementation Schemes 23-26, wherein at least one of steps (b), (c) and (d) is performed on an automated analyzer.

[0236] Illustrative Embodiment 28. The method of Illustrative Embodiment 27, wherein step (c) is further defined as measuring the amount of relative optical units (RLU), wherein the amount of RLU is proportional to the amount of NT-proBNP in the sample.

[0237] Illustrative Implementation 28A. A method of any one of Illustrative Implementations 23-28, wherein the method is performed in an emergency department setting.

[0238] Illustrative Implementation Scheme 28B. The method of any one of Illustrative Implementation Schemes 23-28A, wherein the method is performed in an outpatient setting.

[0239] Illustrative Implementation Scheme 28C. The method of any one of Illustrative Implementation Schemes 23-28, wherein the method is performed in an emergency department and / or outpatient setting.

[0240] Illustrative Embodiment 29. A non-transient computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including any one of the methods in illustrative embodiments 23-28C.

[0241] Illustrative Implementation Scheme 30. A method for determining the presence, severity, and / or susceptibility to heart failure (HF) in an individual, the method comprising the steps of: (a) Combining the following simultaneously or in whole or in part sequentially to form a reaction mixture: (i) Biofluid samples; (ii) a solid-phase reagent comprising a solid support having a first anti-NT-proBNP antibody or a binding fragment thereof to which it is attached; and (iii) A simplified reagent comprising a second anti-NT-proBNP antibody or a binding fragment thereof labeled with acridinium ester, wherein the first and second anti-NT-proBNP antibodies or the binding fragment thereof bind to non-overlapping epitopes of NT-proBNP; (b) Incubate the reaction mixture under conditions that allow (ii) and (iii) to bind with NT-proBNP present in the biofluid 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, wherein the method optionally includes the following steps: (d) Based on the measured value of NT-proBNP in the sample, a mathematical algorithm is used to obtain the HF score, wherein the biofluid sample is selected from blood, serum, plasma and combinations thereof.

[0242] Illustrative Implementation Scheme 31. Implementation Scheme 30 includes the implementation of the methods described herein in emergency department, point-of-care, and / or outpatient settings.

[0243] Illustrative embodiments 32, 30-31, wherein the biofluid sample is obtained in an outpatient setting and subsequently provided to the reaction mixture.

[0244] Illustrative implementation scheme 33, implementation schemes 30-32, wherein the individuals are characterized as being from the outpatient stratum.

[0245] Illustrative embodiments 34, 30-33, wherein the first anti-NT-proBNP antibody or its binding fragment is biotinylated, and wherein at least a portion of the surface of the solid support is coated with streptavidin, thereby indirectly attaching the first anti-NT-proBNP antibody or its binding fragment to the solid support, wherein the solid support comprises magnetic particles and / or latex particles, wherein: (ii) the solid-phase reagent comprises a biotinylated monoclonal sheep anti-human antibody specific for NT-proBNP conjugated to the magnetic particles, the magnetic particles having streptavidin coated on at least a portion of their surface; and (iii) the simplified reagent comprises an acridinium ester-labeled monoclonal sheep anti-human NT-proBNP F(ab')2 fragment specific for NT-proBNP, wherein no biotin interference was observed at biotin concentrations up to about 3510 ng / ml in the biofluid sample, and wherein at least one of steps (b), (c) and (d) is performed on an automated analyzer.

[0246] Therefore, compositions, kits, systems, and / or methods that fully satisfy the objectives and advantages set forth above have been provided in accordance with this disclosure. Although this disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth above, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Accordingly, it is intended to cover all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. A kit for determining the amount of NT-proBNP in a biological sample, the kit comprising: (a) A solid-phase reagent comprising a solid support having a first anti-NT-proBNP antibody or a binding fragment thereof directly or indirectly attached thereto; and (b) A simplified reagent comprising a secondary anti-NT-proBNP antibody or a binding fragment thereof labeled with acridinium ester; and 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.

2. The kit according to claim 1, wherein the first anti-NT-proBNP antibody or its binding fragment is biotinylated, and wherein at least a portion of the surface of the solid support is coated with streptavidin, thereby indirectly attaching 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 kit according to claim 1, wherein: (a) The solid-phase reagent comprises a biotinylated monoclonal sheep anti-human antibody specific for NT-proBNP conjugated to magnetic particles, said magnetic particles having streptavidin coated on at least a portion of their surface; and (b) The simplified reagent contains an acridinium ester-tagged monoclonal sheep anti-human NT-proBNPF(ab')2 fragment that is specific to NT-proBNP.

5. The kit according to claim 1, wherein (a) and (b) are packaged separately.

6. The kit according to claim 1, wherein the kit is configured for emergency department and / or outpatient settings.

7. A method for determining the presence, severity, and / or susceptibility to heart failure (HF) in an individual, the method comprising the steps of: (a) Combining the following simultaneously or in whole or in part sequentially to form a reaction mixture: (i) Biofluid samples; (ii) A solid-phase reagent comprising a solid support having a first anti-NT-proBNP antibody or a binding fragment thereof to which it is attached; as well as (iii) A simplified reagent comprising a second anti-NT-proBNP antibody or a binding fragment thereof labeled with acridinium ester, wherein the first and second anti-NT-proBNP antibodies or the binding fragment thereof bind to non-overlapping epitopes of NT-proBNP; (b) Incubate the reaction mixture under conditions that allow (ii) and (iii) to bind with NT-proBNP present in the biofluid sample to form an immune complex; and (c) Measure the amount of immune complexes formed to obtain a measurement of NT-proBNP in the sample.

8. The method of claim 7, further comprising the following steps: (d) Based on the measured NT-proBNP values ​​in the sample, a mathematical algorithm is used to obtain the HF score.

9. The method of claim 7, wherein the biofluid sample is selected from blood, serum, plasma, and combinations thereof.

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

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

12. The method according to claim 7, wherein: (ii) The solid-phase reagent comprises a biotinylated monoclonal sheep anti-human antibody specific for NT-proBNP conjugated to magnetic particles, said magnetic particles having streptavidin coated on at least a portion of their surface; and (iii) The simplified reagent contains an acridinium ester-tagged monoclonal sheep anti-human NT-proBNP F(ab')2 fragment that is specific to NT-proBNP.

13. The method of claim 7, wherein no biotin interference was observed at biotin concentrations up to about 3510 ng / ml in the biofluid sample.

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

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

16. The method of claim 7, wherein the method is performed in an emergency department, point-of-care, and / or outpatient setting.

17. The method of claim 7, wherein the biofluid sample is obtained in an outpatient setting and subsequently provided to the reaction mixture.

18. The method of claim 7, wherein the individual is characterized as being from the outpatient stratum.

19. A non-transient computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including the method of claim 7.

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