Reference material for biological testing

Abstract

The present invention relates to novel methods, compositions, kits and systems for the sensitive detection of biomarkers in immunoassays. Disclosed herein are reference materials and methods for monitoring the precision and accuracy of various immunodiagnostic testing methodologies in clinical laboratories.

Description

[0001] REFERENCE MATERIAL FOR BIOLOGICAL TESTING

[0002] Field of the Invention

[0003] The invention relates to methods, compositions, kits and systems for the sensitive detection of biomarkers in immunoassays.

[0004] Background to the Invention

[0005] Commercially available quality control or reference materials are routinely used in clinical diagnostics laboratories to monitor the precision and accuracy of both manual and automated clinical test methods and procedures. These controls are typically prepared by spiking various analytes (drugs, hormones, enzymes, antibodies, antigens, etc.) into a base matrix containing various additives such as stabilizers and antimicrobial agents and are offered in liquid and lyophilized formats. Processed human base matrices such as human serum or human urine may be used for manufacturing of quality controls in order to ensure that the controls are as sensitive as the actual patient samples to all anticipated analytical variances.

[0006] Quality controls are typically offered as single analyte or multi-analyte controls in bi-level or tri-level configurations to monitor and challenge the performance of the test methods at above, near, and below the medical decision point for each assay. Typically controls have analyte lists that contain related analytes, for example cardiac markers or analytes measured by one type of detection technology, for example, routine chemistry analytes measured by photometry or urinalysis analytes measured by reflectance photometry using dry chemistry strips. In addition, quality control materials are designed to be stable, provide lot-to-lot reproducibility, and be cost effective.

[0007] A number of high-sensitivity (hs) immunoassays have been developed that can very precisely record slightly elevated and rising concentrations of biomarkers very early after onset of clinical symptoms. These assays have been developed by different manufacturers for specific platforms such as Abbott Architect, Siemens Centaur, Siemens Atellica, Siemens Vista, and Beckman Coulter Access. However, developing a quality control or reference material that could be used on all testing platforms, which have different analytical and performance characteristics, has been a significant technical challenge. This technical challenge has resulted in the introduction of assay-specific or platform-specific reference materials. This lack of standardization and harmonization of high sensitivity assays is a major disadvantage as the results obtained on different platforms may present significant differences making it difficult to compare analytical parameters and clinical performance between tests. Therefore, there is a need for a reference material that is more commutable for immunoassays and provides closer agreement among measurement methodologies.

[0008] It is against this background that the present methods, compositions and systems have been devised.

[0009] Summary of the Invention

[0010] According to a first aspect of the invention, there is provided a reference material for an immunoassay to detect and / or quantify the presence of a first biomarker, said reference material comprising a first antigen at a first concentration and a second antigen at a second concentration, the first and second antigens being derived from and / or corresponding to antigens of the first biomarker.

[0011] The reference material is a quality control material, a calibration standard or proficiency testing material.

[0012] Suitably, the first concentration of the first antigen is different to the second concentration of the second antigen.

[0013] In embodiments, the first and second antigens are naturally derived, synthetically derived, derived by recombination, or a combination thereof. Suitably, the first and second antigens are derived from nonhuman and / or non-native sources.

[0014] In embodiments, the first and second antigens are present in a base matrix. Suitably, the base matrix is, comprises or consists of, human plasma, serum, whole blood, urine, or is a synthetic medium.

[0015] In embodiments, the reference material further comprises a third antigen derived from the first biomarker. Suitably, the concentration of the third antigen is different to the concentrations of the first and second antigens respectively; or the concentration of the third antigen is the same as the concentration of either the first antigen or the second antigen. More suitably, the concentration of the first, second and / or third antigen is selected to be a clinically relevant concentration. Suitably, the concentration of the first, second and / or third antigen is above a predefined threshold.

[0016] In embodiments, the first, second and / or third biomarker is a marker associated with a physiological and / or a pathogenic condition. The biomarker may be selected from one of the group consisting of: a hypertension biomarker, for e.g., a cardiac biomarker, a tumor biomarker, an inflammatory biomarker, a bone turnover biomarker, a sepsis-related biomarker, a hormone biomarker, or a CD biomarker.

[0017] In embodiments, the hypertension marker is selected from the group consisting of ACTH and ADH.

[0018] In embodiments, The cardiac marker is selected from the group consisting of: troponin (e.g. troponin T (nT), troponin I (Tnl), preferably Tn I), CK-MB, myoglobin, BNP and NT-proBNP.

[0019] In embodiments, the tumor marker is selected from the group consisting of: beta-2 microglobulin, ACTH, AFP, CA 15-3, CA 19-9, CA 27-29, CA 50, CA72-4, CA 125, calcitonin, CEA, CASA, Cyfra 21- 1 , ferritin, hCG, HE4, Her2 / neu, IGF-1 , NSE, PAP, Pro GRP, prolactin, PSA, S-100, SCC and thyroglobulin.

[0020] In embodiments, the inflammatory marker is selected from the group consisting of: CRP, hsCRP and IL-6.

[0021] In embodiments, the bone turnover marker is selected from the group consisting of: Osteocalcin, Parathyroid hormone (PTH), and Bone-specific alkaline phosphatase (BALP).

[0022] In embodiments, the sepsis-related marker is selected from the group consisting of: Procalcitonin and C-reactive protein (CRP).

[0023] In embodiments, the hormone marker is a sex hormone marker selected from the group consisting of: estradiol, testosterone, hCG, p-hCG, LH and FSH.

[0024] In embodiments, the hormone marker is a hormone marker selected from the group consisting of: iPTH, calcitonin, ACTH, TSH and osteocalcin.

[0025] In embodiments, the reference material further comprises at least one antigen derived from a second biomarker. Suitably, the second biomarker is a marker associated with the same physiological and / or pathogenic condition as the first biomarker.

[0026] In embodiments, the reference material does not contain serum proteases. Suitably, the reference material is substantially free of proteases. According to a second aspect of the invention, there is provided a method for preparing a reference material for an assay to detect and / or quantify a biomarker, said the method comprising: obtaining a first antigen and a second antigen, the first and second antigens being derived from and / or corresponding to antigens of the biomarker; and mixing the first antigen with the second antigen at different concentrations.

[0027] According to a third aspect of the invention, there is provided a method for diagnosing or prognosing a disease or disorder in a patient, the method comprising: detecting the presence and / or quantifying the amount of a biomarker for the disease or disorder in a sample obtained from the patient; and comparing the detection or amount of the biomarker in the sample with the detection or amount of at least one antigen in a reference material, wherein the detection or amount of the biomarker in the patient sample relative to the detection or amount of the at least one antigen in the reference material indicates the presence, absence, or prognosis of the disease or disorder; and wherein the reference material is according to the first aspect of the invention.

[0028] Optional and preferred features of the first aspect, as set out in the description, clauses and claims apply, unless stated otherwise, to the features of the second and third aspects, and vice versa.

[0029] Brief Description of the Drawings

[0030] Figure 1 shows the recovery of Troponin I in the Abbott Calibrator on various test methods using Siemens Centaur as the anchor test method.

[0031] Figure 2 shows the recovery of Troponin I in a quality control material utilizing recombinant Tnl from Raybiochem on various test methods using Siemens Centaur as the anchor test method.

[0032] Figure 3 shows the recovery of Troponin I in a quality control material utilizing recombinant Tnl from Spectral on various test methods using Siemens Centaur as the anchor test method.

[0033] Figure 4 shows the recovery of Abbott Architect and Siemens Centaur Tnl Calibrators.

[0034] Figure 5 shows the recovery of different recombinant Tnl raw materials on major clinical diagnostic analyzers. Figure 6 shows the commutability of a reference material according to the present invention between different analyzers.

[0035] Detailed Description of the Invention

[0036] The present invention provides a reference material for biological assays, including immunoassays. Reference material as used herein includes quality control material, calibration standard and proficiency testing material.

[0037] Quality control is a vital function of every immunoassay to ensure that the results are within acceptable limits of precision and accuracy. Additionally, for quantification of a target analyte in a sample, it is generally necessary to first establish a calibration curve which represents the relationship between the analytical signal obtained from the particular analytical method used and the quantity of the target analyte. Thus, prior to the analysis of a sample the analytical signals of a series of calibration standards, or calibrators, have to be determined and this external calibration has to be done regularly, e.g., daily. Proficiency testing is a tool for quality assessment and quality control for laboratories and allows for interlaboratory comparison of assay results. Commutability of reference materials is a critical property to ensure standardisation between assays.

[0038] The term “commutability” was first used to describe the ability of a reference or control material for enzyme measurements to have interassay properties comparable to the properties demonstrated by authentic clinical samples when measured by more than one analytical method. ISO 15194 defines commutability as the property of a given reference material demonstrated by the closeness of agreement between the relation among the measurement results for a stated quantity in the material obtained according to two measurement procedures and relation obtained among the measurement results specified material. ISO 17511 defines commutability as closeness of agreement between mathematical relationship of the measurement results obtained by two measurement procedures for a stated quantity in a given material and the mathematical relationship obtained for the quantity in routine samples.

[0039] There are a number of assays in need of standardization. An exemplary assay is the high-sensitivity cardiac Troponin I assay. Other assays include, but are not limited to, assays for the detection of total cholesterol, HDL-cholesterol, LDL-cholesterol, total glycerides, lipoproteins, vitamin D, parathyroid hormone, estradiol, testosterone, sex hormone binding globulin, thyroid hormones, thyroid stimulating hormones, anti-thyroglobulin, anti-thyroperoxidase, BNP, NT-proBNP, ferritin, osteocalcin, intact-PTH, CA 125, CA 15-3 and hCG. High-sensitivity cTnl assay

[0040] Cardiac troponin I (cTnl) is considered the key biomarker for diagnosis of acute myocardial infarction (AMI) and myocardial infarction (Ml). This biomarker is released into the blood stream during a cardiac event. High-sensitivity cTnl tests can be used for rapid triage and early diagnosis or exclusion of Ml. Troponin I testing can be used to rule out Ml and to identify low-risk patients, which in turn results in avoiding unnecessary hospital admissions and improving emergency care. The 99th percentile upperreference limit (URL) of cardiac troponin has served as the assay-specific threshold to diagnose myocardial injury.

[0041] Due to the lack of standardization and harmonization of high sensitivity cardiac Troponin I assays (hs- cTnl), even from the same manufacturer, all assays have different 99th percentiles, analytical parameters, and clinical performance. Thus, commercially available quality control or reference material lacks adequate commutability for all assays.

[0042] Presented in Table 1 are the 99th percentiles for several commercially available hs-cTnl test methods demonstrating poor agreement among the test methods regarding medical decision points.

[0043] Table 1: 99th percentiles for several commercially available hs-cTnl test methods.

[0044] The poor agreement among these test methods can be attributed to the use of different antigen and antibody pairs utilized by different manufacturers in their immunoassay reagent kits. Various raw material manufacturers offer antigens and antibodies that can be used to construct an immunoassay for hs-cTnl testing. For example, HyTest Ltd. (Turku, Finland) offers different antibodies that can be used for quantitative determination of Tnl for immunoassay development with specificity toward epitopes 22-40 (Y306 and Y503), 83-100 (Y101), 161-178 (Y501), and 174-191 (Y502) of Troponin I.

[0045] Figure 1 demonstrates the percent recovery values for the Abbott Architect Calibrator when tested on Abbott Architect, Siemens Centaur, Siemens Atellica, Siemens Vista, and Beckman Coulter Access as a sample using Siemens Centaur as the anchor method (100% recovery). As can be seen, Siemens platforms (Centaur, Atellica, and Vista) show similar recoveries most likely due to similar assay architecture (using the same chemistry and antigen-antibody matched pair). However, recovery of the same Abbott Calibrator (reference material) on different test methodologies demonstrate poor commutability. This figure shows that the antibodies used in the Siemens assays (Centaur, Vista, and Atellica) do not recognize some of the epitopes in the antigen used in the Abbott Calibrator, and therefore, show lower percent recovery versus Abbott Architect.

[0046] Presented in Figure 2 are similar percent recovery values for a quality control material prepared by spiking a recombinant Troponin I raw material (from Ray Biotech Life, Inc., Catalog # 64-2-L4, Peachtree Corners, GA) into human serum when tested on Abbott Architect, Siemens Centaur, Siemens Atellica, Siemens Vista, and Beckman Coulter Access as a sample using Siemens Centaur as the anchor method (100% recovery). As it can be seen, Siemens platforms (Centaur, Atellica, and Vista) show similar percent recoveries most likely due to similar assay architecture as described above; however, recovery of the same quality control material (reference material) on different test methodologies demonstrate poor commutability. Once again, this figure clearly shows that the antibodies used in these assays have different affinities toward the recombinant Troponin I antigen used in the quality control material with Siemens Atellica exhibiting the highest percent recovery and Abbott Architect showing the lowest percent recovery versus the anchor method of Siemens Centaur used in this study.

[0047] Figure 3 demonstrates another study that further exemplifies the problem described in Figures 1 and 2. In this study a quality control material was also prepared by spiking a recombinant Troponin I raw material (from Spectral Medical Inc., Catalog # 3500, Toronto, Canada) into human serum. This quality control was then tested on Abbott Architect, Siemens Centaur, Siemens Atellica, Siemens Vista, and Beckman Coulter Access as a sample using Siemens Centaur as the anchor method (100% recovery). As can be seen, Siemens platforms (Centaur, Atellica, and Vista) show similar percent recoveries; but recovery of the same quality control material (reference material) on different test methodologies demonstrates poor commutability. This figure further supports that the antibodies used in these assays recognize different epitopes on the recombinant Troponin I antigen used as the quality control material, with Siemens Atellica exhibiting the highest percent recovery and Beckman Access showing the lowest percent recovery versus the anchor method of Siemens Centaur used in this study.

[0048] Reference materials

[0049] The present disclosure provides a multi-level quality control, or reference, material, which could be used on multiple assays I platforms and provides Troponin I values, which are consistent and commutable across multiple immunoassays using different antigen-antibody pairs used in the assay architecture. It is envisaged that the reference material disclosed herein may be used as a calibration standard or quality control for use with any analyzer, or with a group of different analysers.

[0050] According to an aspect, the present invention provides a reference material to detect and / or quantify the presence of a first biomarker, said reference material comprising a first antigen at a first concentration and a second antigen at a second concentration, the first antigen and the second antigen being and / or corresponding to antigens of the first biomarker.

[0051] The term “antigen” as used herein refers to any molecule that can bind specifically to an antibody. An antigen is recognized by antibodies, T-cell receptors or other elements of specific humoral and / or cellular immunity. Furthermore, for purposes of the present invention, "antigen" also can be used to refer to a protein that includes modifications, such as deletions, additions and substitutions (generally conservative in nature, but they may be non-conservative), to the native sequence, as long as the protein maintains the ability to elicit an immunological response. The antigen can be derived, obtained, or isolated from a naturally occurring protein or may be synthetically derived. Synthetic antigens include polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens.

[0052] In embodiments, the reference material further comprises a third antigen, the third antigen being and / or corresponding to an antigen of the biomarker.

[0053] In embodiments, the first, second and / or third antigens are different to each other. In this embodiment, the first, second and / or third antigens comprise different epitopes of the antigen of the biomarker.

[0054] In embodiments, the first, second and third antigens are at different concentrations to each other.

[0055] In embodiments, the first, second and / or third antigens are purified antigens.

[0056] In embodiments, the reference material comprises a fourth, a fifth, a sixth antigen and so on, the antigens being and / or corresponding to an antigen of the biomarker. The antigens may be similar to or different from each other.

[0057] In embodiments, the second, third, fourth, fifth, sixth (and so on) antigens are or correspond to antigens of different biomarkers.

[0058] In embodiments, the first and second antigens are a recombinant Troponin I raw material. In embodiments, the first, second and / orthird antigens is selected from: recombinant human Troponin I antigen, recombinant human Troponin I antigen stabilised with recombinant rabbit skeletal muscle troponin-C, recombinant single chain cardiac Troponin l-C (Human cTnl-linker-cTnC).

[0059] In embodiments, the reference material comprises (i) a recombinant human Troponin I antigen precomplexed with recombinant rabbit Troponin C in vitro 6% vol / vol, (ii) a stabilized recombinant human Troponin I antigen 22% vol / vol, and (iii) recombinant Troponin I and C protein subunits expressed in a single polypeptide chain 72% vol / vol.

[0060] In embodiments, the antigens in the reference material are present in a base matrix. Suitable base matrices are known in the art. In embodiments, the base matrix comprises or consists of human plasma, serum, whole blood, urine or is a synthetic medium. In embodiments, matrix constituents include but are not limited to human serum albumin, immunoglobins, other proteins, lipids, salts, stabilizers, various exogenous chemicals including antimicrobials, protease inhibitors and buffers.

[0061] In embodiments, the base matrix is buffered. In embodiments, the pH of the base matrix is adjusted to 6.2.

[0062] The term "biomarker" or “marker” as used herein refers to a biological marker characterizing a phenotype. A biomarker typically includes a gene an mRNA or a protein.

[0063] Examples of such biomarkers include proteins or polypeptides. Proteins or polypeptides used as a biomarker in the present invention are contemplated to include naturally occurring fragments of a protein; in particular, immunologically detectable fragments. Immunologically detectable fragments may comprise at least 6, 7, 8, 10, 12, 15, 20 or more contiguous amino acids of said marker polypeptide.

[0064] Troponin: “Troponin” is a regulatory protein that controls the calcium-mediated interaction of actin and myosin in the contraction of striated muscle. Troponin has three subunits: troponin C, troponin I and troponin T.

[0065] Cardiac troponin (CTn) is troponin that regulates contraction of cardiac muscle. Troponin T and I are unique to cardiac myocytes. As used herein, CTn refers to all Troponin isoforms expressed in cardiac cells, including cardiac Troponin I (cTnl) and cardiac Troponin T (cTnT). CK-MB: "CK-MB" (Muscle-Brain type Creatine Kinase, also referred to as "Creatine Kinase-MB") is a well-known biomarker in the art. It is one of the three isoenzymes of creatine kinase (CK), which is an enzyme expressed by various tissues and cell types, and which catalyses the conversion of creatine using adenosine triphosphate to generate phosphocreatine and adenosine diphosphate. Creatine kinases comprise two subunits - brain type (B) or muscle type (M) subunits. The isoenzyme CK-MB comprises a brain type and a muscle type subunit. The primary source of CK-MB is myocardial. However, it is also found in the skeletal muscle.

[0066] BNP-type peptide: The Brain Natriuretic Peptide type peptide (herein also referred to as "BNP-type peptide") is preferably selected from the group consisting of pre-proBNP, proBNP, NT-proBNP, and BNP. The pre-pro peptide (134 amino acids in the case of pre-proBNP) comprises a short signal peptide, which is enzymatically cleaved off to release the pro peptide (108 amino acids in the case of proBNP). The pro peptide is further cleaved into an N-terminal pro peptide (NT-pro peptide, 76 amino acids in case of NT-proBNP) and the active hormone (32 amino acids in the case of BNP). Preferably, the BNP-type peptide according to the present invention is BNP or, in particular, NT-proBNP. BNP is the active hormone and has a shorter half-life than its respective inactive counterpart NT-proBNP.

[0067] The term “predetermined threshold” refers to a threshold value of antigen(s) in a test sample which, for example, allows for the differentiation between a subject at risk of a physiological or pathological condition and a subject not at risk of a physiological or pathological condition.

[0068] In an embodiment, the physiological condition is myocardial infarction.

[0069] In some embodiments, the threshold concentration of troponin is determined by analyzing samples, e.g., serum, or plasma samples, from an apparently healthy population for cardiac troponin, e.g., cardiac troponin I, and determining the level at which 80, 90, 95, 96, 97, 98, 99, 99.5, or 99.9% of the population fall below that level (concentration). This value is the threshold value. In some embodiments, the threshold value is set at the 99th percentile. In some embodiments, the analyzing is performed using a method with a level of detection for the cardiac troponin of about 5 pg / ml or less, about 3 pg / ml or less, or about 1 pg / ml or less, e.g., less than about 5 pg / ml, or between about 5 pg / ml and 0.5 pg / ml.

[0070] In some embodiments, the invention provides a method for diagnosing predicting or prognosing by comparing a value for a concentration of cardiac troponin in a sample from the individual with a normal value for cardiac troponin, where the normal value is determined by a cardiac troponin assay with a limit of detection for the cardiac troponin in the sample of less than about 50, 40, 30, 10, 5, 4, 3, 2 or 1 pg / ml, e.g., less than about 20 pg / ml; and ii) diagnosing, predicting or prognosing, based on comparison.

[0071] In some embodiments, the reference material according to the present invention is used to detect and / or quantify the presence of one or more tumour biomarkers.

[0072] "Tumor biomarker" or "cancer biomarker" refers to a biomarker characteristic of a tumor or cancer but not normal tissue. Tumor markers envisaged in the present invention include but are not limited to: Beta-2 microglobulin, ACTH, AFP, CA 15-3, CA 19-9, CA 27-29, CA 50, CA 72-4, CA 125, Calcitonin, CEA, CASA, Cyfra 21-1 , Ferritin, hCG, HE4, Her2 / neu, IGF-1 , NSE, PAP, Pro GRP, Prolactin, PSA, S-100, SCC and Thyroglobulin.

[0073] In embodiments, the reference material according to the present invention is used to detect and / or quantify the presence of one or more cardiac biomarkers.

[0074] In embodiments, the reference material according to the present disclosure is used to detect and / or quantify the presence of one or more inflammatory biomarkers. Inflammatory biomarkers include but are not limited to: CRP, hsCRP, and IL6.

[0075] In embodiments, the reference material according to the present disclosure is used to detect and / or quantify the presence of one or more hypertension biomarkers. Hypertension biomarkers include but are not limited to: adrenocorticotropic hormone (ACTH) and antidiuretic hormone (ADH).

[0076] In embodiments, the reference material according to the present disclosure is used to detect and / or quantify the presence of one or more reproductive health biomarkers. Reproductive health biomarkers include but are not limited to estradiol, testosterone, hCG, p-hCG, LH and FSH.

[0077] In embodiments, the reference material according to the present invention is used to detect and / or quantify the presence of one or more hormone biomarkers. Hormone biomarkers include but are not limited to iPTH, Calcitonin, ACTH, TSH and Osteocalcin

[0078] Clinically relevant concentrations

[0079] In embodiments, the reference material according to the present invention comprises a first, a second and / or a third antigen at a clinically relevant concentration, i.e., a physiological or a pathological concentration. Clinically relevant concentrations corresponding to normal ranges for different biomarkers are shown in Table 2.

[0080] In embodiments where the first, second or third biomarker is a bone turnover biomarker, the biomarker is present in the range of 14-46 ng / mL.

[0081] In embodiments where the first, second or third biomarker is a sepsis-related biomarker, the biomarker is present in the range of <0.05 ng / mL.

[0082] In embodiments where the first, second or third biomarker is a hormone biomarker, the biomarker is present in the range of 3-16 ng / dL. In embodiments where the first, second or third biomarker is a cardiac biomarker, the biomarker is present in the range of <0.03 ng / mL. In embodiments where the first, second or third biomarker is a hypertension biomarker, the biomarker is present in the range of 1 .0-3.0 pg / mL.

[0083] In embodiments where the first, second or third biomarker is an inflammatory biomarker, the biomarker is present in the range of <1.0 mg / L.

[0084] The "subject" as referred to herein may be a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, pigs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats and guinea pigs). Suitably, the subject is a human.

[0085] The term "sample" refers to a blood, serum or plasma sample. The sample may be prepared in any appropriate way; for example, it may be frozen, fresh, fixed (e.g. formalin fixed), centrifuged, and / or embedded (e.g. paraffin embedded), as known to the person skilled in the art.

[0086] The term “pathological condition” as used herein refers to any disease, disorder or condition that is to be treated or receive a treatment.

[0087] The terms "disorder" and "disease" are used interchangeably herein and comprise any undesired physiological or pathological change in a subject, an animal, an isolated organ, a tissue or a cell I cell culture.

[0088] Method for diagnosis or prognosis

[0089] In an aspect, the present disclosure provides a method for diagnosing or prognosing a disease or disorder in a patient, the method comprising detecting the presence of and I or quantifying the amount of a biomarker for the disease or disorder in a sample obtained from the patient. Suitably, the method comprises an immunoassay. The reference material or quality control material used in the assay is beneficially as described herein.

[0090] Individuals who will benefit from the present methods may be exhibiting symptoms of an acute or chronic disease. Alternatively, the subject may be suspected of having experienced an acute or chronic disease. Examples of acute diseases include but are not limited to transient ischemic attack (TIA) or stroke. Examples of chronic diseases include, but are not limited to, cardiovascular disease such as atherosclerosis; inflammatory disorders such as Crohn’s disease, inflammatory bowel disease; hypertension and cancer. In some embodiments, the levels of expression of the panel of biomarkers is determined within 3 hours of onset of a suspected acute disease. In some embodiments, the levels of expression of the panel of biomarkers are determined at 3 or more hours after onset of a suspected acute disease. In some embodiments, the levels of expression of the panel of biomarkers are determined within 6, 12, 18, 24, 36, or 48 hours of onset of a suspected acute disease.

[0091] In some cases, the subject is asymptomatic, but may have a risk or predisposition to experiencing an acute or chronic disease, e.g., based on genetics, a related disease condition, environment or lifestyle. For example, in some embodiments, the patient may suffer from a chronic inflammatory condition, e.g., has an autoimmune disease (e.g., rheumatoid arthritis, Crohn's disease inflammatory bowel disease), atherosclerosis, hypertension, or diabetes. In embodiments, the patient has high LDL- cholesterol levels or suffers from a cardiovascular disease (e.g., atherosclerosis, coronary artery disease). In embodiments, the patient has an endocrine system disorder, a neurodegenerative disorder, a connective tissue disorder, or a skeletal and muscular disorder.

[0092] The terms "treat" or "treatment" as used herein refer to therapeutic treatment as well as prophylactic or preventative measures, wherein the object is to prevent or reduce the progression of an undesired physiological or pathological change or disorder. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the manifestation of the condition or disorder is to be prevented.

[0093] Kits

[0094] In an aspect, the present disclosure provides kits for performing an assay, including an immunoassay, comprising a reference material as disclosed herein. Reagents, tools, and I or instructions for performing the methods described herein can be provided in a kit. For example, the kit can contain reagents, tools, and instructions for detecting and I or quantifying a biomarker. The biomarker may be a cardiac biomarker, tumor biomarker, hypertension biomarker, inflammatory biomarker, sepsis-related biomarker, hormone biomarker, bone turnover biomarker, or other physiologically, pathogenically and I ortherapeutically-relevant biomarker. In a specific embodiment, the biomarker is a cardiac biomarker.

[0095] In some instances, the kits may also contain the specific therapeutic agent to be administered in the event that the test detects or predicts a physiologic or pathologic condition. This agent may be provided in any suitable form, such as a dosage form, that is tailored to the specific treatment. The kit may be provided with suitable instructions for administration according to an appropriate treatment regimen. This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof.

[0096] Examples

[0097] Example 1: Recovery of Abbott Architect and Siemens Centaur Tnl Calibrators

[0098] Presented in Figure 4 are the recovery values for commercially available and dedicated calibrators for Abbott Architect and Siemens Centaur when tested on these two test methods. As can be seen in Figure 4, when Abbott Architect calibrator is tested on Siemens Centaur, the recovery value is less than 50% of the concentration measured on Abbott Architect. The opposite is observed when Siemens Centaur calibrator is tested on Abbott Architect. In this case, the recovery value on Abbott Architect is less than 50% of the Tnl concentration measured on Siemens Centaur. This study suggests that different Tnl raw materials are used to manufacture these calibrators, and - as would be expected - the antibodies used in the assays have stronger affinity for their intended matched antigens.

[0099] Combining these two calibrators that use different Tnl raw materials at 35% Abbott Calibrator and 65% Siemens Calibrator (vol / vol) resulted in a mixture that when spiked into a buffered base matrix, produced compositions with varying concentrations of Tnl and produced test results that are in close agreement with each other when tested on both Siemens Centaur and Abbott Architect (Table 3).

[0100] Table 3: Agreement of test results when the stock solution is used to prepare compositions (dilutions) with concentration of Tnl below and near the medical decision point.

[0101] Example 2: Recovery of different recombinant Tnl raw materials on major clinical diagnostic analyzers

[0102] Presented in Figure 5 are the recovery values for 3 different recombinant Troponin I raw materials from 3 different suppliers (Biospacific, Inc., Emeryville, CA, Life Diagnostics, Inc., West Chester, PA, and Spectral Medical Inc., Toronto, Canada). To prepare samples forthis study, Siemens Centaur was used as the anchor test method and sufficient Tnl raw materials from different suppliers were measured and spiked into a buffered base matrix (consisting of 20% HSA, pH = 7.2) to prepare high concentration Tnl stock solutions containing 10,000 to 16,000 pg / mL. As can be seen in Figure 5, these recombinant Tnl raw materials show different recovery values on different test methods. For example, Tnl from Life Diagnostics demonstrates the highest recovery on Siemens Centaur and the lowest recovery on Abbott Architect (Figures 5a); and Tnl from Spectral shows the highest recovery on Siemens Centaur and lowest recovery on Beckman Access (Figure 5c).

[0103] Example 3: A composition containing various percentages of multiple Tnl raw materials to achieve male 99th percentile targets on multiple analyzers

[0104] To demonstrate the applicability of the invention, a mixture was prepared by blending stock solutions containing Tnl. The stock solutions that underwent blending were formulated from commercially available Troponin I antigen raw materials in liquid form with added buffers and preservatives with the following general characteristics and blend percentage utilization: (i) a recombinant human Troponin I antigen pre-complexed I stabilized with a separate source of recombinant rabbit Troponin C in vitro 6% vol / vol, (ii) a stabilized recombinant human Troponin I antigen 22% vol / vol, and (iii) both recombinant Troponin I and C protein subunits expressed in a single polypeptide chain 72% vol / vol. This multi-raw material high concentration spiking mixture was then spiked into the buffered processed serum base matrix to prepare a composition with low concentration of Tnl (at approximately 99th percentile for the male population) for Siemens Centaur, Abbott Architect, and Beckman Access. The results can be seen in Figure 6. This study clearly demonstrates that a single QC composition can be prepared that meets the target requirements for male 99th percentile for different test methods using different raw materials with different recovery characteristics.

[0105] Clauses

[0106] 1. A reference material for an immunoassay to detect and / or quantify the presence of a first biomarker, said reference material comprising a first antigen at a first concentration and a second antigen at a second concentration, the first and second antigens being derived from and / or corresponding to antigens of the first biomarker.

[0107] 2. The reference material according to Clause 1 , wherein the first concentration of the first antigen is different to the second concentration of the second antigen.

[0108] 3. The reference material according to Clause 1 or Clause 2, wherein the first and second antigens are naturally derived, synthetically derived, derived by recombination, or a combination thereof.

[0109] 4. The reference material according to any preceding clause, wherein the first and second antigens are derived from non-human and / or non-native sources. 5. The reference material according to any preceding clause, wherein the first and second antigens are present in a base matrix.

[0110] 6. The reference material according to Clause 5, wherein the base matrix is, comprises or consists of human plasma, serum, whole blood, urine, or a synthetic medium.

[0111] 7. The reference material according to Clause 5 or Clause 6, wherein the base matrix is buffered.

[0112] 8. The reference material according to any preceding clause, further comprising a third antigen derived from the first biomarker.

[0113] 9. The reference material according to any preceding clause, further comprising a third antigen derived from the first biomarker, and wherein the third antigen has a third concentration.

[0114] 10. The reference material according to Clause 9, wherein the concentration of the third antigen is different to the concentrations of the first and second antigens respectively; or wherein the concentration of the third antigen is the same as either the first concentration or the second concentration.

[0115] 11. The reference material according to any preceding clause, wherein the concentration of the first and / or second antigen is selected to be a clinically relevant concentration.

[0116] 12. The reference material of Clause 9, or Clause 10 or Clause 11 when dependent on Clause 9, wherein the concentration of the third antigen is selected to be a clinically relevant concentration.

[0117] 13. The reference material of Clause 11 or Clause 12, wherein the concentration of the first, second and / or third antigen is above a predefined threshold.

[0118] 14. The reference material of any of Clauses 11 to 13, wherein the concentration of the first, second and / or third antigen is selected to be suitable for detecting or diagnosing a physiological condition.

[0119] 15. The reference material according to any preceding clause, wherein the reference material is a control, a calibrator, a standard or a proficiency material. 16. The reference material according to any preceding clause, wherein the first biomarker is a marker associated with a physiological and / or a pathogenic condition.

[0120] 17. The reference material according to any preceding clause, wherein the first, second and / or third biomarker is a marker associated with a physiological and / or pathogenic condition, and wherein the first biomarker is selected from one of the group consisting of: a hypertension marker, a cardiac marker, a tumor marker, an inflammatory marker, a bone turnover marker, a sepsis-related marker, a hormone marker, or a CD marker.

[0121] 18. The reference material according to Clause 17, wherein the hypertension marker is selected from the group consisting of ACTH and ADH.

[0122] 19. The reference material according to Clause 17, wherein the cardiac marker is selected from the group consisting of: troponin (e.g. troponin T, troponin I), CK-MB, myoglobin, BNP and NT- proBNP.

[0123] 20. The reference material according to Clause 17, wherein the tumor marker is selected from the group consisting of: beta-2 microglobulin, ACTH, AFP, CA 15-3, CA 19-9, CA 27-29, CA 50, CA 72-4, CA 125, calcitonin, CEA, CASA, Cyfra 21-1 , ferritin, hCG, HE4, Her2 / neu, IGF-1 , NSE, PAP, Pro GRP, prolactin, PSA, S-100, SCC and thyroglobulin.

[0124] 21 . The reference material according to Clause 17, wherein the inflammatory marker is selected from the group consisting of: CRP, hsCRP and IL-6

[0125] 22. The reference material according to Clause 17, wherein the bone turnover marker is selected from the group consisting of: Osteocalcin, Parathyroid hormone (PTH), and Bone-specific alkaline phosphatase (BALL).

[0126] 23. The reference material according to Clause 17, wherein the sepsis-related marker is selected from the group consisting of: Procalcitonin and C-reactive protein (CRP).

[0127] 22. The reference material according to Clause 17, wherein the hormone marker is a sex hormone marker selected from the group consisting of: estradiol, testosterone, hCG, p-hCG, LH and FSH. 23. The reference material according to Clause 17, wherein the hormone marker is a hormone marker selected from the group consisting of: iPTH, calcitonin, ACTH, TSH and osteocalcin.

[0128] 24. The reference material according to any preceding clause, further comprising at least one antigen derived from a second biomarker.

[0129] 25. The reference material according to any preceding clause, wherein the reference material does not contain serum proteases.

[0130] 26. The reference material according to any preceding clause, wherein the reference material is substantially free of proteases.

[0131] 27. The reference material according to Clause 24, wherein the second biomarker is a marker associated with the same physiological and / or pathogenic condition as the first biomarker.

[0132] 28. A method for preparing a reference material for an assay to detect and / or quantify a biomarker, the method comprising: obtaining a first antigen and a second antigen, the first and second antigens being derived from and / or corresponding to antigens of the biomarker; and mixing the first antigen with the second antigen at different concentrations.

[0133] 29. The method of Clause 29, wherein the reference material is defined according to any of Clauses 1 to 28.

[0134] 30. A kit for performing an assay to detect and / or quantify a biomarker, the kit comprising a reference material according to any of Clauses 1 to 28.

[0135] 31. A method for diagnosing or prognosing a disease or disorder in a patient, the method comprising: detecting the presence and / or quantifying the amount of a biomarker for the disease or disorder in a sample obtained from the patient; and comparing the detection or amount of the biomarker in the sample with the detection or amount of at least one antigen in a reference material, wherein the detection or amount of the biomarker in the patient sample relative to the detection or amount of the at least one antigen in the reference material indicates the presence, absence, or prognosis of the disease or disorder; and wherein the reference material is a reference material according to any of Clauses 1 to 28. 32. The method according to Clause 31 , wherein detecting the amount of the biomarker comprises determining the concentration of the biomarker. 33. The method of Clause 31 or Clause 32, wherein the at least one antigen of the reference material corresponds to at least one antigen of the biomarker.

Claims

CLAIMS:

1. A reference material for an immunoassay to detect and / or quantify the presence of a first biomarker, said reference material comprising a first antigen at a first concentration and a second antigen at a second concentration, the first and second antigens being derived from and / or corresponding to antigens of the first biomarker.

2. The reference material according to Claim 1 , wherein the first concentration of the first antigen is different to the second concentration of the second antigen.

3. The reference material according to Claim 1 or Claim 2, wherein the first and second antigens are naturally derived, synthetically derived, derived by recombination, or a combination thereof.

4. The reference material according to any preceding claim, wherein the first and second antigens are derived from non-human and / or non-native sources.

5. The reference material according to any preceding claim, wherein the first and second antigens are present in a base matrix.

6. The reference material according to Claim 5, wherein the base matrix is, comprises or consists of human plasma, serum, whole blood, urine, or a synthetic medium.

7. The reference material according to Claim 5 or Claim 6, wherein the base matrix is buffered.

8. The reference material according to any preceding claim, further comprising a third antigen derived from the first biomarker.

9. The reference material according to any preceding claim, further comprising a third antigen derived from the first biomarker, and wherein the third antigen has a third concentration.

10. The reference material according to Claim 9, wherein the concentration of the third antigen is different to the concentrations of the first and second antigens respectively; or wherein the concentration of the third antigen is the same as either the first concentration or the second concentration.11 . The reference material according to any preceding claim, wherein the concentration of the first and / or second antigen is selected to be a clinically relevant concentration.

12. The reference material of Claim 9, or Claim 10 or Claim 11 when dependent on Claim 9, wherein the concentration of the third antigen is selected to be a clinically relevant concentration.

13. The reference material of Claim 11 or Claim 12, wherein the concentration of the first, second and / or third antigen is above a predefined threshold.

14. The reference material of any of Claims 11 to 13, wherein the concentration of the first, second and / or third antigen is selected to be suitable for detecting or diagnosing a physiological condition.

15. The reference material according to any preceding claim, wherein the reference material is a control, a calibrator, a standard or a proficiency material.

16. The reference material according to any preceding claim, wherein the first biomarker is a marker associated with a physiological and / or a pathogenic condition.

17. The reference material according to any preceding claim, wherein the first, second and / or third biomarker is a marker associated with a physiological and / or pathogenic condition, and wherein the first biomarker is selected from one of the group consisting of: a hypertension marker, a cardiac marker, a tumor marker, an inflammatory marker, a bone turnover marker, a sepsis-related marker, a hormone marker, or a CD marker.

18. The reference material according to Claim 17, wherein the hypertension marker is selected from the group consisting of ACTH and ADH.

19. The reference material according to Claim 17, wherein the cardiac marker is selected from the group consisting of: troponin (e.g. troponin T, troponin I), CK-MB, myoglobin, BNP and NT-proBNP.

20. The reference material according to Claim 17, wherein the tumor marker is selected from the group consisting of: beta-2 microglobulin, ACTH, AFP, CA 15-3, CA 19-9, CA 27-29, CA 50, CA 72-4, CA 125, calcitonin, CEA, CASA, Cyfra 21-1 , ferritin, hCG, HE4, Her2 / neu, IGF-1 , NSE, PAP, Pro GRP, prolactin, PSA, S-100, SCC and thyroglobulin.

21. The reference material according to Claim 17, wherein the inflammatory marker is selected from the group consisting of: CRP, hsCRP and IL-622. The reference material according to Claim 17, wherein the bone turnover marker is selected from the group consisting of: Osteocalcin, Parathyroid hormone (PTH), and Bone-specific alkaline phosphatase (BALL).

23. The reference material according to Claim 17, wherein the sepsis-related marker is selected from the group consisting of: Procalcitonin and C-reactive protein (CRP).

24. The reference material according to Claim 17, wherein the hormone marker is a sex hormone marker selected from the group consisting of: estradiol, testosterone, hCG, p-hCG, LH and FSH.

25. The reference material according to Claim 17, wherein the hormone marker is a hormone marker selected from the group consisting of: iPTH, calcitonin, ACTH, TSH and osteocalcin.

26. The reference material according to any preceding claim, further comprising at least one antigen derived from a second biomarker.

27. The reference material according to any preceding claim, wherein the reference material does not contain serum proteases.

28. The reference material according to any preceding claim, wherein the reference material is substantially free of proteases.

29. The reference material according to Claim 26, wherein the second biomarker is a marker associated with the same physiological and / or pathogenic condition as the first biomarker.

30. A method for preparing a reference material for an assay to detect and / or quantify a biomarker, the method comprising: obtaining a first antigen and a second antigen, the first and second antigens being derived from and / or corresponding to antigens of the biomarker; and mixing the first antigen with the second antigen at different concentrations.31 . The method of Claim 30, wherein the reference material is defined according to any of Claims 1 to 29.

32. A kit for performing an assay to detect and / or quantify a biomarker, the kit comprising a reference material according to any of Claims 1 to 29.

33. A method for diagnosing or prognosing a disease or disorder in a patient, the method comprising: detecting the presence and / or quantifying the amount of a biomarker for the disease or disorder in a sample obtained from the patient; and comparing the detection or amount of the biomarker in the sample with the detection or amount of at least one antigen in a reference material, wherein the detection or amount of the biomarker in the patient sample relative to the detection or amount of the at least one antigen in the reference material indicates the presence, absence, or prognosis of the disease or disorder; and wherein the reference material is a reference material according to any of Claims 1 to 28.

34. The method according to Claim 33, wherein detecting the amount of the biomarker comprises determining the concentration of the biomarker.

35. The method of Claim 33 or Claim 34, wherein the at least one antigen of the reference material corresponds to at least one antigen of the biomarker.

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