Use of spermidine for analyzing proteins in heparin-treated samples

Abstract

The present invention concerns the field of diagnostics. In particular, it relates to a method for analyzing a protein analyte in a heparin-treated sample comprising or suspected to comprise said protein analyte, said method comprising the step of contacting said heparin-treated sample with spermidine prior to analyzing the said protein analyte in the said sample. Moreover, contemplated are a method for assessing a cardiovascular disease or disorder, a neurological disease or disorder, a metabolic disease or disorder or cancer as well as kits and reagent compositions comprising spermidine to be used in the said methods.

Description

[0001] Roche Diagnostics GmbH RD39759PC AD / JK

[0002] Use of spermidine for analyzing proteins in heparin-treated samples

[0003] The present invention concerns the field of diagnostics. In particular, it relates to a method for analyzing a protein analyte in a heparin-treated sample comprising or suspected to comprise said protein analyte, said method comprising the step of contacting said heparin-treated sample with spermidine prior to analyzing the said protein analyte in the said sample. Moreover, contemplated are a method for assessing a cardiovascular disease or disorder, a neurological disease or disorder, a metabolic disease or disorder or cancer as well as kits and reagent compositions comprising spermidine to be used in the said methods.

[0004] Heparin in sample tubes can interfere with heterogeneous or immunoassays in general through various mechanisms, including aggregation, binding to proteins, antibody formation, enzyme inhibition, alteration of the sample matrix, and impact on signal generation. Understanding these interferences and implementing appropriate mitigation strategies is essential for obtaining accurate and reliable assay results. There are five possible mechanisms how heparin can interfere not only in heterogeneous immunoassays but also in all other types of assays. These mechanisms can be but are not limited to:

[0005] Heparin-Induced Aggregation: Heparin can cause the aggregation of assay components, such as beads or particles used in the immunoassay. This aggregation can interfere with the proper binding and separation steps required for accurate measurement. Aggregation can lead to false positive or false-negative results by either trapping the analyte or preventing it from binding to the detection antibodies. The likelihood of occurrence is low, as heparin is typically not in the assay formulation.

[0006] Binding to Proteins: Heparin has a strong negative charge and can bind to positively charged proteins, especially with antigens, e.g. troponins measured in these tests (Tate, J. & Ward, G. Interferences in immunoassay. Clin. Biochem. Rev. 25, 105-20 (2004)). This binding can block the interaction sites on the antibodies or antigens, reducing the assay's sensitivity, specificity, and recovery to serum. It can also alter the conformation of these proteins, affecting their functionality. The likelihood of occurrence is high, as troponins and other highly positively charged proteins can be affected.

[0007] Heparin-Induced Antibody Formation: In some individuals, heparin can induce the formation of antibodies against heparin-protein complexes (heparin-induced thrombocytopenia or HIT antibodies). These antibodies can interfere with immunoassays by cross-reacting with assay components, leading to erroneous results. The likelihood of occurrence is medium, as this has been described in the literature (Lee, G. M. & Arepally, G. M. Heparin-induced thrombocytopenia. Hematology 2013, 668-674 (2013); Linkins, L. A., Bates, S. M., Lee, A. Y. Y. & Warkentin, T. E. Combination Of 4T’s Score and Rapid Gel Centrifugation Assay Excludes HIT In a Prospective Cohort Study. Blood 122, 3535 (2013)) but only occurs when patients have been injected with heparin, e.g. after an acute myocardial infarction (AMI) and develop HIT antibodies.

[0008] Interference with Enzyme Activity: Heparin can inhibit the activity of enzymes used in certain immunoassays, such as enzyme-linked immunosorbent assays (ELISA) or others. Inhibition of enzyme activity can reduce the signal generation, leading to underestimation of the analyte concentration. The likelihood of occurrence is low, as this has only been reported for enzymatic immunoassays yet, which is not the case within Elecsys heterogeneous assays as no enzymatic reaction is used but can occur in other heterogeneous assays (Armstrong, G. P., Barker, A. N., Patel, H. & Hart, H. H. Reference interval for troponin I on the ACS: Centaur assay: a recommendation based on the recent redefinition of myocardial infarction. Clin. Chem. 48, 198-9 (2001); Squires, M., Wise, H., Holmes, H. & Hadfield, K. Lithium heparin interference in the Abbott enzymatic creatinine assay: the significance of under-filled tubes. Ann. Clin. Biochem. 58, 653-656 (2021); Yip, P. M., Chan, M. K., Zielinski, N. & Adeli, K. Heparin interference in whole blood sodium measurements in a pediatric setting. Clin. Biochem. 39, 391-395 (2006)).

[0009] Alteration of Sample Matrix: Heparin can alter the overall composition of the sample matrix. This can affect the assay's performance by changing the binding dynamics or the stability of the assay components. Changes in the sample matrix can lead to variability in the assay results, reducing the reliability and reproducibility of the measurements. The likelihood of occurrence is high, as we have shown this for troponin assays before and used interference eliminating substances before such as polybrene.

[0010] Especially for Troponin assays, it is known that the second and fifth mechanism have an impact on assay quality and reproducibility in comparison with other sample types such as serum or EDTA. Specifically, troponins are overall positively charged proteins that might cause interference with strongly negatively charged heparin. Thus, it is of importance to reduce this interaction by assay components. Currently, polybrene (hexadimethrine bromide), which is a positively charged polymer, is used in the Troponin T assays on the market. Polybrene is often used to neutralize the anticoagulant effects of heparin in blood samples. Heparin is a negatively charged molecule, and polybrene, being positively charged, can bind to heparin and neutralize its effects. Drawbacks of polybrene are a high lot-to-lot variation, there are restrictions for the use of polybrene as a harmful chemical, in particular, in light of oral toxicity, for some biomarker assays, polybrene promotes cross-reactivity.

[0011] There is a need for improving the assay quality of protein biomarker assays such as the Troponin T assay avoiding the drawbacks of using polybrene.

[0012] The technical problem underlying the present invention may be seen as the provision of means and methods complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

[0013] The present invention relates to a method for analyzing a protein analyte in a heparin-treated sample comprising or suspected to comprise said protein analyte, said method comprising the step of contacting said heparin-treated sample with spermidine prior to analyzing the said protein analyte in the said sample.

[0014] It is to be understood that in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "an" item can mean that at least one item can be utilized.

[0015] As used in the following, the terms "have", "comprise" or "include" may be meant to have a non-limiting meaning or a limiting meaning. Thus, having a limiting meaning these terms may refer to a situation in which, besides the feature introduced by these terms, no other features are present in an embodiment described, i.e. the terms have a limiting meaning in the sense of "consisting of' or "essentially consisting of'. Having a non-limiting meaning, the terms refer to a situation where besides the feature introduced by these terms, one or more other features are present in an embodiment described.

[0016] Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "typically", and "more typically" or similar terms are used in conjunction with additional or alternative features, without restricting alternative possibilities. Further, it will be understood that the term "at least one" as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one item shall be used this may be understood as one item or more than one item, i.e. two, three, four, five or any other number. Depending on the item the term refers to the skilled person understands as to what upper limit the term may refer, if any.

[0017] The method according to the present invention may consist of the aforementioned step or may comprise further steps, such as pretreating or isolating the sample prior to carrying out the recited step and / or after the step one or more steps of evaluating the obtained result of the analysis.

[0018] The term "analyzing" as used herein refers to determining the presence or absence or the abundance of the protein analyte in the sample. Said determination may, thus, be a qualitative determination, i.e. the determination of the presence or absence, or a quantitative determination, i.e. the determination of the abundance. The determination may be made by measuring at least one physicochemical property of the protein analyte or at least one biological property of the protein analyte qualitatively or quantitatively. The physicochemical property of the protein analyte may be any physical or chemical property which allows for determining the protein analyte. Preferably, said property may be a molecular weight, spectroscopic property, immunological property (binding specificity), or the like. A biological property may be any biological activity elicited by the protein analyte, preferably, an enzymatic activity, cell activation activity or the like.

[0019] Determination may be carried out directly or indirectly. Direct measuring relates to measuring the amount or concentration of the protein analyte based on a signal which is obtained from the protein analyte molecule itself and the intensity of which directly correlates with the number of molecules of the protein analyte present in the sample. Such a signal - sometimes referred to herein as intensity signal - may be obtained, e.g., by measuring an intensity value of a specific physical or chemical property of the biomarker molecule. Indirect measuring includes measuring of a signal obtained from a secondary component, i.e. a component not being the protein analyte molecule itself.

[0020] In accordance with the present invention, determining the amount of a protein analyte can be achieved by all known means for determining such amounts in a sample. Said means comprise immunoassay devices and methods which may utilize detection agents such as labeled molecules in various sandwich, competition, or other assay formats. Said assays will develop a signal which is indicative for the presence or absence of the protein analyte. Moreover, the signal strength can, preferably, be correlated directly or indirectly (e.g. reverse- proportional) to the amount of the protein analyte present in a sample. Further suitable methods comprise measuring a physical or chemical property specific for the protein analyte. Said methods comprise, preferably, biosensors, optical devices coupled to immunoassays, biochips, or other analytical devices such as chromatography devices or single cell analyzing devices such as FACS.

[0021] Preferably, for determining a protein analyte in accordance with the invention, a binding agent is applied that specifically binds to the said protein analyte and that can be detected either by a detectable label present in the detection agent or by a secondary binding molecule that specifically binds to the detection agent and comprises a detectable label.

[0022] The term "binding agent" as referred to in this context may be any molecule that is capable of specifically binding to the protein analyte to be detected. Preferably, such a binding agent is selected from the group consisting of antibodies, aptamers, darpins, affibodies, and analyte binding proteins, such as protein or peptide receptors or ligands.

[0023] An antibody in accordance with the present invention may encompass all types of antibodies which specifically bind to the biomarker protein. Preferably, the antibody of the present invention is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody or any fragment or derivative of such antibodies being still capable of binding to the biomarker protein specifically. Such fragments and derivatives comprised by the term antibody as used herein encompass a bispecific antibody, a synthetic antibody, a Fab, F(ab)2 Fv or scFv fragment, or a chemically modified derivative of any of these antibodies. Specific binding as used in the context of the antibody of the present invention means that the antibody does not cross react with other molecules present in the sample to be investigated. Specific binding can be tested by various well-known techniques. Antibodies or fragments thereof, in general, can be obtained by using methods which are described in standard text books, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals and, preferably, immunized mice.

[0024] An antibody mimetic in accordance with the present invention encompasses peptide or protein molecules that have antibody-like binding properties but which are not structurally related to antibodies. Such antibody mimetics have typically a molecular weight of up to 20 kDa. Preferably, an antibody mimetic in accordance with the present invention may be an affibody molecule, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, an darpin, a fynomer, a gastrobody, a Kunitz domain protein, a monobody, a nanoCLAMP, a repedody, a centryn or an obody.

[0025] An aptamer according to the present invention may be a nucleic acid or peptide aptamer. Specific aptamers can be generated by techniques well known in the art including, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) technology. Peptide aptamers comprise of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint shall increase the binding affinity of the peptide aptamer into the nano-molar range. Said variable peptide loop length is, typically, composed of ten to twenty amino acids, and the scaffold may be any protein having improved solubility and compacity properties, such as thioredoxin-A. Peptide aptamer selection can be made using different systems including, e.g., the yeast two-hybrid system. The term also encompasses optimized or modified aptamers such as optimers, split aptamers or X-aptamers.

[0026] A detectable label as referred to herein which may be used in accordance with the invention include gold particles, latex beads, acridinium ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels, e.g., magnetic beads, including paramagnetic and superparamagnetic labels, and fluorescent labels. Enzymatically active labels include e.g. horseradish peroxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'- tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3- indolyl-phosphate. A suitable enzyme-substrate combination may result in a colored reaction product, fluorescence or chemiluminescence, which can be measured according to methods known in the art (e.g. using a light-sensitive film or a suitable camera system). As for measuring the enzymatic reaction, the criteria given above apply analogously. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes. Also the use of quantum dots as fluorescent labels is contemplated. Typical radioactive labels include 35S, 1251, 32P, 33P and the like. A radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager. Suitable labels may also be or comprise tags, such as biotin, digoxygenin, His-, GST-, FLAG-, GFP-, MYC-tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like.

[0027] The term "protein analyte" as used herein refers to a protein comprised in the sample or being suspected to be comprised in the sample. Accordingly, it will be understood that the protein analyte may be any protein that naturally occurs in the sample material. E.g., if blood is used as a sample material, the protein analyte may be any protein that naturally occurs in blood. A protein as referred to herein also encompasses peptides. Peptides are proteins that typically consist of less amino acids, e.g., less than about 100 amino acids. Protein analytes which are naturally present in sample material envisaged in accordance with the present invention are described elsewhere herein in more detail.

[0028] Preferably, said protein analyte is a positively-charged protein or peptide. Preferably, there are criteria for determining whether a protein or peptide is whether a positively-charged protein or peptide as referred to herein. The criteria are discussed in the following and a positively-charged protein or peptide shall in accordance with the present invention fulfill at least one, at least two, at least three or all four of these criteria.

[0029] First, positively-charged proteins or peptides in accordance with the present invention, preferably, have a higher isoelectric point (pl). The pl indicates the pH at which a protein carries no net charge. Proteins with a higher isoelectric point are more positively charged at physiological pH. Higher pl values indicate a greater positive charge under physiological conditions. More preferably, the pl of a positively charged protein according to the invention is at pH 7.0, 7.5, 8.0, 8.5 or 9.0.

[0030] Second, positively-charged proteins or peptides in accordance with the present invention, preferably, have a higher proportion of basic amino acids, i.e. lysine, arginine, and / or histidine, in their amino acid sequences. A higher content of these amino acids contributes to a higher overall positive charge. Typically, a higher content as meant herein refers to an occurrence of the basic amino acids in percent (%) which is above the average for said amino acids found in proteins. The average is for lysine about 7.0%, for arginine about 4.7% and for histidine about 2.1%.

[0031] Third, positively-charged proteins or peptides in accordance with the present invention, preferably, have a higher positive charge density, i.e. a higher regional distribution of positive charges along the protein's surface. Proteins with a high density of positive charges in specific regions can interact more strongly with negatively charged molecules.

[0032] Fourth, positively-charged proteins or peptides in accordance with the present invention, preferably, may in its natural environment biologically interact with negatively-charged molecules such as DNA, RNA or membranes or may be involved in the antimicrobial response of an organism.

[0033] Preferably, the protein analyte to be analyzed in accordance with the present invention is a biomarker indicative for a disease or disorder in a subject. A "biomarker" as used in accordance with the present invention relates to the biomarker protein or any precursor protein, fragment or derivative thereof which is naturally generated and which reflects the amount of the biomarker protein.

[0034] More preferably, said positively-charged protein or peptide is cardiovascular protein biomarker. Most preferably, said cardiovascular biomarker is selected from the group consisting of a cardiac troponin, preferably troponin T or troponin I, a natriuretic peptide, preferably, NT- proBNP, pro BNP, BNP, or ANP, C-reactive protein (CRP), interleukin 6 (IL-6), an apolipoprotein and lipoprotein (a). Most preferably, the cardiovascular biomarker in accordance with the present invention is a cardiac troponin and, preferably, troponin T.

[0035] The Table indicate the abbreviations of the biomarkers, their full designation and an UniProt accession number for each biomarker which, preferably, shows the human amino acid sequence of the biomarker protein.

[0036] Table: Cardiovascular protein biomarkers

[0037] More preferably, said positively-charged protein or peptide is a neurological biomarker protein. Most preferably, said neurological biomarker is selected from the group consisting of: Neurofilament (NFL), Tau, preferably, pTau217 or pTaul81, Neurotrophin (3 or 4), Myelin Basic Protein (MBP) or Synuclein.

[0038] Table: Neurological biomarker protein

[0039] More preferably, said positively-charged protein or peptide is a metabolic or cancer biomarker protein. Most preferably, said metabolic or cancer biomarker is selected from the group consisting of C reactive protein (CRP), IL-6, TNF-alpha, VEGF, an apolipoprotein, Transferrin, Ferritin, Insulin, Glucagon, PTH or Calcitonin.

[0040] Table: Metabolic or cancer biomarker protein It will be understood that biomarkers as referred to herein, in addition to those biomarkers having the specific sequence as deposited under the UniProt accession number also encompass variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned proteins deposited under the respective UniProt accession numbers. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino sequence of the biomarker protein as deposited under the indicated UniProt accession number, preferably over the entire length of the said biomarker protein, respectively.

[0041] The degree of identity between two amino acid sequences in accordance with the present invention can be determined by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm disclosed by Smith, by the homology alignment algorithm of Needleman, by the search for similarity method of Pearson, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. Variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs. Variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs.

[0042] Fragments of the aforementioned biomarkers may also be determined in accordance with the present invention. The term "heparin treated sample" as used herein a sample of, preferably, a body fluid, which needs to be treated by heparin. Blood samples, e.g., are treated by heparin in order to avoid coagulation. More preferably, said sample is, thus, a blood, serum or plasma sample.

[0043] The term "contacting" as used herein refers to physically contacting spermidine to the sample and the protein analyte comprised therein or suspected to be comprised therein as well the heparin comprised by the sample. In particular, but without being bound by theory, it is envisaged that spermidine molecules can interact with heparin molecules in the sample such that the protein analyte molecules are released from or prevented from molecular interactions with heparin molecules.

[0044] Contacting can be, preferably, done by admixing a composition comprising spermidine with the sample. Alternatively, contacting may be done by dissolving spermidine in the sample. Accordingly, it will be understood that spermidine is upon contacting present in the heparin- treated sample, preferably, in at least in an equimolar amount to heparin. More preferably, spermidine may be present in excess with respect to heparin. The skilled person is well aware of how respective solutions of spermidine can be provided or how spermidine can be dissolved in such desired amount in heparin-treated samples. Most preferably, spermidine can be used as described in the accompanying examples, below.

[0045] Upon contacting the sample with spermidine in accordance with the method of the present invention, preferably, the protein analyte present in the heparin-treated sample is recovered by at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%. Recovering the protein analyte to this extent from heparin, more preferably, will help to avoid false classifications of samples due to heparin-bound or heparin-inactivated protein analyte which is not available for the analysis. For example, if a protein analyte is bound to heparin it may not be properly detected by an antibody or it may not properly exert its enzymatic activity. As a consequence, the amount of the protein analyte determined in the sample may be below a certain threshold although its true amount is above that threshold. As a result the sample may be characterized as negative although being positive, i.e. false negative.

[0046] The term "spermidine" as used herein refers to a polyamine compound having the formula C7H19N3 and the UP AC designation N1-(3-Aminopropyl)butane-l,4-diamine. Spermidine has been registered under CAS 124-20-9 (free base) or CAS 334-50-9 (trihydrochloride). Spermidine is, preferably, used in solution in the method of the present invention. Typically, a spermidine solution comprising excessive spermidine with respect to the heparin in the sample is used. Preferably, such a solution is an aqueous solution comprising spermidine in a concentration as to provide a final concentration in the sample upon admixing of between about 10 and about 30 mM / L, preferably, between about 15 and about 25 mM / L, more preferably about 17 mM / L, about 18 mM / L, about 19 mM / L or about 20 mM / L. In case spermidine is dissolved as solid matter, it is envisaged that upon dissolving the spermidine as, e.g., suitable salt, in a liquid sample, spermidine is present in a concentration of between about 10 and about 30 mM / L, preferably, between about 15 and about 25 mM / L, more preferably about 17 mM / L, about 18 mM / L, about 19 mM / L or about 20 mM / L. Particular preferred solutions comprising spermidine which may be applied in accordance with the method of the present invention are also described in the examples, below. In general, the effectiveness of spermidine in neutralizing heparin typically depends on the concentration of both spermidine and heparin, as well as the specific conditions of the assay. While spermidine might neutralize heparin, it could also interact with other negatively charged molecules in the sample or assay reagents, potentially leading to non-specific effects. The concentration and conditions under which spermidine is added can be optimized by the skilled artisan to ensure it effectively neutralizes heparin without adversely affecting the assay.

[0047] Advantageously, it has been found in the studies underlying the present invention that adding Spermidine to heparin-treated samples allows for superior analytic results, in particular, when protein analytes and typically positively charged protein analytes are investigated. Spermidine is a positively charged polyamine, and effectively eliminating interference in diagnostic assays, particularly those affected by negatively charged molecules like heparin. By neutralizing heparin's negative charges, spermidine prevents heparin-induced aggregation and inhibition of assay components, ensuring more accurate and reliable results. Additionally, spermidine enhances binding interactions between assay components; increases assay sensitivity and specificity, and stabilizes proteins, leading to consistent and reproducible outcomes. Its use can be optimized to avoid adverse effects, making spermidine a valuable alternative to other positively charged molecules like polybrene used in current commercial biomarker assays. Overall, spermidine improves diagnostic performance and patient outcomes by mitigating assay interference while being available at reproducible quality and quantity for scalable production of diagnostics tests.

[0048] Surprisingly, Spermidine turned out to be a superior alternative compared to polybrene or other potential substitutes. Without being bound by theory, it is assumed that Spermidine neutralizes charge, i.e. Spermidine is a positively charged molecule, which means it can interact with and neutralize negatively charged molecules like heparin. By binding to heparin, spermidine could reduce its negative charge, potentially mitigating its interference in the assay. interacts with heparin, i.e. the multiple positive charges on spermidine could allow it to bind effectively to the negatively charged sulfate groups on heparin, thereby neutralizing its charge and reducing its impact on the assay components.

[0049] Spermidine does not exhibit a high lot-to-lot variability. Moreover, it is a non-toxic and environmental safe chemical. Further, for proteins such as cardiac troponins and, in particular, cardiac troponin T, no cross reactivity with skeletal muscle troponin T was observed under some assay conditions (see Examples below). Accordingly, Spermidine shall be superior over polybrene in avoiding cross-reactivity of protein analytes.

[0050] The present invention further relates to a method for assessing a cardiovascular disease or disorder comprising the steps of: a) analyzing a protein analyte by the method of the invention for analyzing a protein analyte in a heparin-treated sample; and b) assessing the cardiovascular disease or disorder based on the analyzed protein analyte.

[0051] The term "assessing" as used herein refers to diagnosing a disease or disorder referred to herein, e.g., a cardiovascular disease or disorder, neurological disease or disorder or a metabolic disease or disorder or a cancer, in a subject, differentiating between different types of said disease, or predicting said disease or disorders in a subject. As will be understood by those skilled in the art, an assessment is usually not intended to be correct for 100% of the subjects to be investigated. The term, however, requires that the assessment is correct for a statistically significant portion of the subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.

[0052] Assessing as referred to herein, preferably, comprises comparing the analyzed protein, preferably, the quantitatively or qualitatively analyzed protein analyte with a reference. The reference shall allow for assessing the subject (from which the sample that has been analyzed for the protein analyte by the method of the invention) as suffering from a disease or disorder referred to herein, or not (rule-in or rule-out diagnosis), predicting the risk for developing a disease or disorder referred to herein, or not, (rule-in or rule out prediction) or differentiating one of the disease or disorders from others referred to herein. It will be understood that a reference as referred to herein relates to any amount or value which by comparison to the determined amount of the protein analyte (biomarker) allows for assessing the disease or disorder in the subject. Thus, the reference amount or value may be obtained from a subject or a group of subjects known to suffer from a disease or disorder referred to herein. In such a case, if similar amounts for the biomarkers are present in the test sample, the tested subject shall be assessed as suffering from the disease or disorder, whereas if amounts for the biomarkers which differ from the reference are determined in the test sample, the subject may be assessed as not suffering from said disease or disorder. The reference amount or value may also be obtained from a subject or a group of subjects known not to suffer from a disease or disorder referred to herein. In such a case, if similar amounts for the biomarkers are present in the test sample, the tested subject shall be assessed as not suffering from the disease or disorders, whereas if amounts for the biomarkers which differ from the reference are determined in the test sample, the subject may be assessed as suffering from said diseases or disorder. The same applies mutatis mutandis if assessing relates to predicting the risk for a disease or disorder referred to herein or differentiating one disease or disorder from others.

[0053] The term "cardiovascular diseases or disorders" as used herein refers to diseases and disorders affecting the heart and / or blood vessels or leading to impaired circulation and various complications. These disorders can result from various factors, including lifestyle, genetic factors, or other underlying health conditions. Preferably, a cardiovascular disease or disorder as referred to herein is selected from the group consisting of coronary artery diseases, preferably, angina pectoris, myocardial infarction, acute coronary syndrome, or silent ischemia, heart failure, preferably, congestive heart failure, right or left sided heart failure or biventricular heart failure, arrhythmia, preferably, bradycardia, tachycardia, atrial or ventricular fibrillation, primary, secondary or pulmonary hypertension, valve diseases, peripheral artery diseases, preferably, atherosclerosis, carotid artery diseases, peripheral artery diseases, dilated, hypertrophic or restrictive cardiomyopathy, congenital heart diseases, ischemic stroke, hemorrhagic stroke, transient ischemic attack, thrombosis, aneurysm, endocarditis, pericardial diseases, and cardiac arrest. More preferably, the cardiovascular disease or disorder in accordance with the present invention is myocardial infarction.

[0054] Typically, the methods for assessing a disease or disorder referred to herein are to be carried out for a subject being an animal, preferably a farming animal, such as cow, sheep, goat, pig or horse, a pet, such as a cat, dog or bird, or a laboratory animal, such as a monkey, mouse, rat or rabbit. More preferably the subject is a human. The subjects being assessed by the herein described methods may or may not exhibit further symptoms indicative for one or more diseases or disorders to be assessed and, thus, may or may not be suspected already to suffer from a disease or disorder or to be at risk for doing so. Thus, assessing the cardiovascular disease or disorder based on the analyzed protein analyte in step b) of the above method may comprise the steps of: comparing the amount or value determined for the analyzed protein in step a) to a reference; and assessing the cardiovascular disease or disorder based on said comparison.

[0055] The present invention contemplates a method for assessing a neurological disease or disorder comprising the steps of: a) analyzing a protein analyte by the method of the invention for analyzing a protein analyte in a heparin-treated sample; and b) assessing the neurological disease or disorder based on the analyzed protein analyte.

[0056] The term "neurological diseases or disorders" as used herein refers to a disease or disorder which affects the brain, the spinal cord and / or peripheral nerves. These diseases or disorders, typically, impair motor control, cognition, sensation, and autonomic functions. Preferably, the neurological disease or disorder in accordance with the present invention is selected from the group consisting of: dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple system atrophy (MSA), Huntington's disease, progressive supranuclear palsy (PSP), corticobasal degeneration, spinocerebellar ataxia, stroke, epilepsy, seizure disorders, peripheral neuropathy, Guillain-Barre syndrome, Charcot-Marie-Tooth disease, myasthenia gravis, multiple sclerosis (MS), neuromyelitis optica (NMO), acute disseminated encephalomyelitis (ADEM), dystonia, essential tremor, restless leg syndrome, Tourette syndrome, brain tumors, migraine, cluster headache, meningitis, encephalitis, prion protein diseases, cerebral palsy, spinal muscular atrophy (SMA), attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), schizophrenia, obsessive-compulsive disorder (OCD), fragile X syndrome, and stress disorders, and depression.

[0057] Assessing the neurological disease or disorder based on the analyzed protein analyte in step b) of the above method may comprise the steps of: comparing the amount or value determined for the analyzed protein in step a) to a reference; and assessing the neurological disease or disorder based on said comparison.

[0058] The present invention further relates to a method for assessing a metabolic disease or disorder or a cancer comprising the steps of: a) analyzing a protein analyte by the method of the invention for analyzing a protein analyte in a heparin-treated sample; and b) assessing the metabolic disease or disorder or a cancer based on the analyzed protein analyte.

[0059] The term "metabolic disease or disorder" as used herein refers to a disease or disorder affecting the metabolism of an organism. Such diseases or disorder, typically, affect organs of the gastrointestinal system, the liver, the pancreas, the kidney or endocrine organs. More specifically, a metabolic disease or disorder referred to herein is selected from the group consisting of: diabetes type 1 or 2, gestational diabetes, metabolic syndrome, obesity, metabolic myopathies, cystic fibrosis, lysosomal storage disease, hypermetabolism, hyperthyroidism, hypothyroidism, phenylketonuria, galactosemia, tyrosinemia, homocystinuria, cystinuria, urea cycle disorders, mucopolysaccharidoses, glycogen storage diseases, Mitochondrial diseases, Cushing's Syndrome, adrenal insufficiency, congenital adrenal hyperplasia, hyperparathyroidism, hypoparathyroidism, hyperlipidemia, hyperglycemia and hypoglycemia.

[0060] The term "cancer" as used herein refers to a diseases characterized by uncontrolled cell growth and / or migration behavior. A cancer in accordance with the present invention may be a solid tumor cancer or a hematopoietic cancer affecting blood cells or precursors thereof. A cancer in accordance with the present invention may be selected from the group consisting of: adenocarcinoma, squamous cell carcinoma, sarcoma, leukemia, lymphoma, multiple lymphoma, melanoma, basal cell carcinoma, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, gallbladder cancer, small intestinal cancer, anal cancer, lung cancer, prostate cancer, bladder cancer, renal cancer, testicular cancer, breast cancer, thyroid cancer, adrenal cancer, ovarian cancer, cervical cancer, head and neck cancer, glioblastoma, astrocytoma, meningioma, neuroblastoma and oligodendroglioma.

[0061] Assessing the metabolic disease or disorder or a cancer based on the analyzed protein analyte in step b) of the above method may comprise the steps of: comparing the amount or value determined for the analyzed protein in step a) to a reference; and assessing the metabolic disease or disorder or a cancer based on said comparison.

[0062] In general, the present invention provides the use of spermidine for analyzing a protein analyte in a heparin-treated sample comprising or suspected to comprise the protein analyte. Yet, the present invention relates to a kit for carrying out the methods of the invention comprising: i) spermidine; and ii) at least one binding agent which specifically binds to the said protein analyte selected from the group consisting of antibodies, aptamers, darpins, affibodies, and analyte binding proteins, such as protein or peptide receptors or ligands.

[0063] The term "kit" as used herein refers to collection of the aforementioned components, typically, provided separately or within a single container. The container also typically comprises instructions for carrying out the method of the present invention. These instructions may be in the form of a manual or may be provided by a computer program code which is capable of carrying out or supports the determination of the biomarkers referred to in the methods of the present invention when implemented on a computer or a data processing device. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device or may be provided in a download format such as a link to an accessible server or cloud.

[0064] Moreover, the kit may usually comprise reference and / or calibration amounts for the protein analytes as described elsewhere herein in detail. The kit according to the present invention may also comprise further components which are necessary for carrying out the method of the invention such as solvents, buffers, washing solutions and / or reagents required for detection.

[0065] Moreover, the present invention contemplates a reagent composition for carrying out the methods of the invention comprising spermidine.

[0066] The term "reagent composition" as used herein refers to a composition comprising Spermidine and at least one further ingredient required for carrying out the method of the present invention. Such ingredients include buffer substances, stabilizers and further auxiliary compounds. Moreover, the reagent composition, typically, comprises a compound required to specifically analyze a protein analyte of interest such as a binding agent as defined elsewhere herein.

[0067] Thus, preferably, said composition further comprises at least one binding agent which specifically binds to the said protein analyte selected from the group consisting of antibodies, aptamers, darpins, affibodies, and analyte binding proteins, such as protein or peptide receptors or ligands. The following are preferred embodiments envisaged in accordance with the present invention.

[0068] Embodiment 1 : A method for analyzing a protein analyte in a heparin-treated sample comprising or suspected to comprise said protein analyte, said method comprising the step of contacting said heparin-treated sample with spermidine prior to analyzing the said protein analyte in the said sample.

[0069] Embodiment 2: The method of embodiment 1, wherein said sample is a blood, serum or plasma sample.

[0070] Embodiment 3 : The method of embodiment 1 or 2, wherein said analyzing of the protein analyte is carried out by using at least one binding agent which specifically binds to the said protein analyte selected from the group consisting of antibodies, aptamers, darpins, affibodies, and analyte binding proteins, such as protein or peptide receptors or ligands.

[0071] Embodiment 4: The method of any one of claims 1 to 3, wherein a protein analyte present in the heparin-treated sample is recovered by at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%.

[0072] Embodiment 5: The method of any one of embodiments 1 to 4, wherein said protein analyte is a positively-charged protein or peptide.

[0073] Embodiment 6: The method of embodiment 5, wherein said positively-charged protein or peptide is cardiovascular protein biomarker.

[0074] Embodiment 7: The method of embodiment 6, wherein said cardiovascular biomarker is selected from the group consisting of: a cardiac troponin, preferably troponin T or troponin I, a natriuretic peptide, preferably, NT-proBNP, pro BNP, BNP, or ANP, C-reactive protein (CRP), interleukin 6 (IL-6), an apolipoprotein and lipoprotein (a).

[0075] Embodiment 8: The method of embodiment 5, wherein said positively-charged protein or peptide is a neurological biomarker protein.

[0076] Embodiment 9: The method of embodiment 8, wherein said neurological biomarker is selected from the group consisting of Neurofilament (NFL), Tau, preferably, pTau217 or pTaul81, Neurotrophin (3 or 4), Myelin Basic Protein (MBP) or Synuclein. Embodiment 10: The method of embodiment 5, wherein said positively-charged protein or peptide is a metabolic or cancer biomarker protein.

[0077] Embodiment 11: The method of embodiment 10, wherein said metabolic or cancer biomarker is selected from the group consisting of: C reactive protein (CRP), IL-6, TNF-alpha or VEGF, apolipoprotein, transferrin, ferritin, insulin, glucagon, PTH or calcitonin.

[0078] Embodiment 12: A method for assessing a cardiovascular disease or disorder comprising the steps of: a) analyzing a protein analyte by the method of any one of embodiments 1 to 7; and b) assessing the cardiovascular disease or disorder based on the analyzed protein analyte.

[0079] Embodiment 13 : A method for assessing a neurological disease or disorder comprising the steps of: a) analyzing a protein analyte by the method of any one of embodiments 1 to 5 or 8 or 9; and b) assessing the neurological disease or disorder based on the analyzed protein analyte.

[0080] Embodiment 14: A method for assessing a metabolic disease or disorder or a cancer comprising the steps of: a) analyzing a protein analyte by the method of any one of embodiments 1 to 5 or 10 or 11 ; and b) assessing the metabolic disease or disorder or a cancer based on the analyzed protein analyte.

[0081] Embodiment 15: Use of spermidine for analyzing a protein analyte in a heparin-treated sample comprising or suspected to comprise the protein analyte.

[0082] Embodiment 16: A kit for carrying out the method of any one of embodiments 1 to 14 comprising: i) spermidine; and ii) at least one binding agent which specifically binds to the said protein analyte selected from the group consisting of: antibodies, aptamers, darpins, affibodies, and analyte binding proteins, such as protein or peptide receptors or ligands.

[0083] Embodiment 17: A reagent composition for carrying out the method of any one of embodiments 1 to 14 comprising spermidine. Embodiment 18: The reagent composition of claim 17, wherein said composition further comprises at least one binding agent which specifically binds to the said protein analyte selected from the group consisting of antibodies, aptamers, darpins, affibodies, and analyte binding proteins, such as protein or peptide receptors or ligands.

[0084] All references cited throughout this specification are herewith incorporated by reference with respect to the specifically mentioned disclosure content as well as in their entireties.

[0085] FIGURES

[0086] Fig- 1 : Serum with either a known concentration of native 14 ng / L or 300 ng / L cTnT or cTnT depleted serum and then spiked with rec. cTnT either 14 ng / L or 300 ng / L is prepared and used as baseline to calculate the recovery of heparinized tubes. Then either 8 mL, 4mL or 1 mL of this predefined serum is filled into LiHep tubes to generate a lx, 2x or 8x concentration of LiHep in the sample.

[0087] Fig. 2: Recovery of LiHep samples to untreated, serum samples. The recovery in % to untreated serum samples is shown using native samples (black bars) or spiked with rec. cTnT (grey bars) with increasing concentrations of polybrene as interference eliminating agent.

[0088] Fig. 3: Recovery of native cTnT in the presence of skTnT. The recovery in % to a serum sample without skTnT is shown using native samples spiked with rec. fast skTnT (black bars), spiked rec. slow skTnT (grey bars) or spiked native skTnT (white bars) containing a mixture of fast and slow skTnT. Increasing concentrations of polybrene as interference eliminating agent are shown.

[0089] Fig. 4: Susceptibility of fragments to heparin interference. Full length (FL), cTnT Fragment 1 and Fragment2. All fragments were used at concentrations of 14 ng / L or 300 ng / L with either the normal concentration of LiHep in the sample or 2x concentration of LiHep. Shown is the recovery of cTnT to a sample without LiHep. Rec. cTnT full length and the slightly shorter fragment 1 showed recoveries below 80%. Fragment 2; 5c) without the C-Terminus was not susceptible to heparin interference.

[0090] Fig. 5: Heparin binding protein (HBP) as possible Interference eliminating agent. Different concentrations of LiHep were simulated with concentrations of 14 ng / L and 300 ng / L rec. cTnT, by filling spiked serum either 4 mL (2x concentration) or 1 mL (8x concentration) into LiHep tubes. The addition of heparin binding protein (HBP) with increasing concentrations did not show beneficial effects for TnThs Gen6 assay formulations with HBP as interference eliminating protein.

[0091] Fig- 6 : Other reagents as possible interference eliminating agent. Different concentrations of LiHep were simulated with concentrations of 14 ng / L and 300 ng / L rec. cTnT, by filling spiked serum either 4 mL (2x concentration) or 1 mL (8x concentration) into LiHep tubes. The addition of ammonium chloride (NH4C1) or histamine didn't show beneficial effects in TnThs (Gen6) assay formulation as interference eliminating agent. Spermidine showed a recovery within the acceptance criteria of 10%, with very good recovery in the 2x concentrated LiHep sample at both concentrations.

[0092] Fig- 7 : Titration of spermidine. Different concentrations of LiHep were simulated with concentrations of 14 ng / L and 300 ng / L rec. cTnT, by filling spiked serum either 8 mL (lx concentration), 4 mL (2x concentration) or 1 mL (8x concentration) into LiHep tubes. The recovery in lx and 2x LiHep for TnThs (Gen6) with 20 mM / L is within acceptance criteria except for very high concentrations of LiHep (8x). Spermidine showed a recovery within the acceptance criteria of 10%, with very good recovery in the 2x concentrated LiHep sample at both concentrations.

[0093] Fig- 8 : Titration of spermidine. Different concentrations of Li-Hep were simulated with concentrations of native, 71 ng / L (Figure A) and rec. cTnl , 74 ng / L (Figure B), by filling serum either 8 mL (lx concentration), 4 mL (2x concentration) or 1 mL (8x concentration) into LiHep tubes. The recovery in lx, 2x and 8x Li-Hep for native serum measured with Tnlhs is within acceptance by using 3.7 mM / L of spermidine up to tested 28 mM / L (A). Using rec. cTnl serum, the recovery in lx, 2x and 8x Li-Hep is with and without spermidine within acceptance criteria.

[0094] Fig. 9 : Titration of spermidine in low, medium and high concentrations of native cTnl. Different concentrations of Li-Hep were simulated with concentrations of native 32.5 ng / L (low), 1345 ng / L (med) and 4861 ng / L (high) concentrated cTnl, by filling serum either 8 mL (lx concentration) (A) or 4 mL (2x concentration) (B) into Li-Hep tubes. In the absence of spermidine, the low and medium samples are not within the acceptance criteria in both concentrations of LiHep. The recovery in lx and 2x Li-Hep for Tnlhs with all tested concentrations of spermidine is within acceptance criteria for low, med and high cTnl concentrated samples (A, B). EXAMPLES

[0095] The Examples merely illustrate the invention. They shall not be construed as limiting the scope.

[0096] Example 1: Identification of interference caused by Lithium Heparin (LiHep) sample type

[0097] In general, throughout the document, it is refered to TnThs (Gen5) to the current on market assay (TnThs in RoW, TnT Gen5 in USA) and TnThs Gen6 to the assay that is in development. If not stated otherwise, TnThs Gen6 refers to an assay formulation that is not used with LiHep interference eliminating agent and may be added or not for the experiment. The artificial experimental setup to test for LiHep interference that is used throughout the development phase to estimate the effects of LiHep interference is described here briefly and throughout the document.

[0098] To test the influence of LiHep the setup is as follows: Serum with known concentration of native cTnT (14 ng / L or 300 ng / L) or troponin depleted serum spiked with full length recombinant cTnT (rec. cTnT) is used if not stated otherwise (e.g. fragments of rec. cTnT are used that are shorter). This is our baseline (or reference) and is used in most experiments to calculate the recovery of heparinized samples. The concentrations used were chosen based on the 99th percentile for TnThs (Gen5) being 14 ng / L and 300 ng / L were chosen to represent a sample with high concentration. This serum is then used and filled with either 8 mL into a LiHep tube (according to the manufacturer's instruction), 4 mL or 1 mL creating a lx, 2x or 8x concentration of LiHep respectively. This setup is schematically drawn in Figure 1. This is an artificial setup to test the influence of LiHep in different settings with changing interference eliminating (IE) agents.

[0099] First 420 serum-LiHep plasma pairs were screened and calculated the number of discrepant results based on strict acceptance criteria, namely if a sample deviates from the serum baseline measured value by more than 10% we count this samples as "discrepant". These results are summarized in Table 1.

[0100] Table 1: Need for improved interference elimination for TnT hs Gen6 420 serum-plasma pairs were measured on cobas e801, STAT application. Acceptance criteria: recovery of counts + / -

[0101] 10% In summary, there is a need to LiHep interference elimination to reduce false-negative recovery of LiHep plasma samples. The susceptibility towards heparin interference seems to be donor dependent or blood draw specific. Susceptible samples and additional samples have been identified and isolated to be used in later experiments (see Table 2 at the end).

[0102] In another experiment, either a native pool of human serum (14 ng / L (Fig. 2A) and 300 ng / L (Fig. 2B) or a depleted serum (2 lots, one lot shown) and spiked with rec. cTnT (14 ng / L (Fig. 2A) and 300 ng / L (Fig. 2B) were used. These native or rec. cTnT pools were then filled in a LiHep tube using 4 mL of the previously generated serum, simulating under filled tubes with 2x concentration of LiHep in the samples (as described in the experimental setup above).

[0103] With this experiment, it was possible to calculate the recovery of these artificial 2x LiHep samples to serum, setting the concentration of serum to 100% and shown in Figure 2 A,B. Additionally, increasing amounts of polybrene were added as interference eliminating agent, with 0.1 g / L being the concentration used in the on-market cTnThs (Gen5). Without the addition of polybrene the recovery of the native sample at 14 ng / L or 300 ng / L was within + / - 10% of the acceptance criteria. In contrast, the recovery of rec. cTnT without polybrene fell to 68%, and 53% respectively for TnThs Gen6.

[0104] Native samples thus seem less sensitive to 2x heparin treatment, while rec. cTnT was more susceptible to heparin interference. The addition of polybrene in the reagent pack was able to eliminate the interference Gen6 assay at similar concentrations of 0.025 g / L polybrene. Increasing amounts of polybrene for Gen6 increased the signal of samples that were spiked with rec. cTnT but had no effect on native samples.

[0105] Additionally, it was tested whether the cross reactivity to skeletal Troponin T isoforms fast (fast skTnT) and slow (slow skTnT) was influenced by increasing amounts of polybrene (in the absence of LiHep). Serum with 9 ng / L native cTnT and spiked in an excess amount of 500 ng / mL (500000 ng / L) of either rec. fast skeletal, rec. slow skeletal or native skeletal TnT (containing a mixture of fast and slow skTnT) was used. In the absence of polybrene there was little cross reactivity to skTnT but increased dramatically after the addition of polybrene in every condition for fast skTnT and native skTnT but not slow skTnT (Fig. 3). The increase in native skTnT is likely due to containing a mixture of both fast and slow skeletal TnT.

[0106] In summary, increasing concentrations of polybrene showed a dramatic increase of crossreactivity to fast and native skTnT when using TnThs (Gen6). Thus, a need for a new interference eliminating agent was of importance for the new assay generation. Example 2: Analysis of the C-terminal part of cTnT as potential root cause for LiHep interference

[0107] In Example 1, a stronger impact of LiHep was noticed on the rec. cTnT spiked in samples. From the literature, it was known that cTnT exists in three fragments in the blood. Fragment 1 : full length cTnT around 40 kDa, fragment 2: cTnT around 29 kDa and fragment 3: around 14-18 kDa. Depending on the time of onset of myocardial infarction, it is known that around 25 % exist as the long form 3 hours after onset, while 15 hours after onset only the smaller fragments 2 and 3 dominate (Cardinaels, E. P. et al. Time-Dependent Degradation Pattern of Cardiac Troponin T Following Myocardial Infarction. Clin. Chem. 59, 1083-1090 (2013)). The TnThs (Gen5) and Gen6 recognizes all fragments that are present in the blood of donors or patients. This was analyzed in more detail by Vxlegzhanina et al. (Vylegzhanina, A. V. et al. Full-Size and Partially Truncated Cardiac Troponin Complexes in the Blood of Patients with Acute Myocardial Infarction. Clin. Chem. 65, 882-892 (2019)) where they used different antibody combinations to identify fragments using size exclusion chromatography (SEC). They could show that cTnT is present in 3 different main forms in blood of patients with different onsets of myocardial infarction or diseases (Vylegzhanina, A. V. et al. Full-Size and Partially Truncated Cardiac Troponin Complexes in the Blood of Patients with Acute Myocardial Infarction. Clin. Chem. 65, 882-892 (2019)).

[0108] Based on Example 1 that rec. cTnT full-length is more susceptible to LiHep interference and based on the literature, that most of the cTnT in patient samples is degraded from the C-terminal end it was hypothesized that only the full-length cTnT is susceptible to heparin interference and thus the root cause (not shown).

[0109] Heparin or LiHep has the highest negative charge density of any know biological molecule. Thus we modelled the electric charge distribution of full length cTnT using Pymol and identified a more negatively charged N-Terminus (not shown) and a more positively charged C-Terminus (not shown) that could possibly interact with negatively charged heparin.

[0110] To test the hypothesis, rec. cTnT fragments were generated using expression constructs generating two fragments of rec. cTnT with either a slightly shorter C-terminus (Fragment 1; Trop2428) or a fragment that has no C-Terminal part (Fragment 2; 5c). All fragments were spiked into troponin free serum at 14 ng / L and 300 ng / L. This was used as 100% and recovery was calculated to the spiked serum without LiHep. We then filled either 4mL (2x LiHep) or 1 mL (8x LiHep) of each spiked serum into a LiHep tube and let it roll for 15 minutes. The recovery of cTnT to spiked serum without LiHep was measured and calculated. Both, the full length and the slightly shortened fragment 1 (Trop2428) were susceptible to LiHep interference and had recoveries below 80%. Fragment 2 (Fig. 4c) did not show interference to LiHep and recovery was 100% of the original value without LiHep (Fig. 4).

[0111] Example 3: Testing of alternative interference elimination agents

[0112] Since polybrene caused a dramatic increase of cross reactivity to skeletal TnT (see Fig. 3) it was set out to test alternative interference elimination agents. Heparin binding protein (HBP), ammonium chloride, histamine, poly-D-lysin (not shown), protamine sulfate (not shown) and spermidine.

[0113] It was tested whether heparin binding protein (HBP) with increasing concentrations could serve as interference eliminating protein. Again, serum was spiked with full-length rec. cTnT at two concentrations (14 ng / L and 300 ng / L) and used as a reference (without LiHep) and either filled 4 mL of this serum into a LiHep tube (2x concentration) or ImL of this serum into a LiHep tube (8x concentration). In the absence of heparin binding protein (HBP) the recovery was between 71 and 80 % and the addition of HBP did not show beneficial effects as interference eliminating protein, even reduced the recovery further.

[0114] Subsequently, ammonium chloride (NH4CI), histamine and spermidine at 20mM / L were tested with the same experiment mentioned above. Without any interference, eliminating agent (IE agent) recovery of rec. cTnT was around 80%. For ammonium chloride (NH4CI), the recovery stayed the same as without. The addition of histamine even lowered the recovery of cTnT. On the other hand, spermidine showed a recovery of up to 90% and better in all conditions tested (Fig. 5, very right).

[0115] More important, there was no increased cross-reactivity to fast skTnT (not shown, recovery to unspiked "empty" fast skTnT was 165%).

[0116] In Table 1, 420 LiHep-serum pairs were tested and 31 interference samples were identified that consistently showed a LiHep interference even in the presence of interference eliminating agents. Those specific samples were tested to analyze whether the addition of spermidine in the assay formulation can significantly reduce the number of discrepant results (Table 2). The addition of spermidine to the assay formulation showed a 4.5 fold reduction of discrepant results, according to our acceptance criteria that the recovery should be with + / - 10% (Table 2). Table 2: Measurement of native interference samples

[0117] 31 serum-plasma pairs identified in Table 1 to be native interference samples were measured on cobas e801, STAT application. Acceptance criteria: recovery of counts + / - 10%.

[0118] At last, different concentrations of spermidine were tested in the assay formulation ranging from ImM / L to 20mM / L. Again cTnT depleted serum was used and spiked in either 14 ng / L or 300 ng / L rec. cTnT. This spiked serum was used as reference and recovery was calculated to the reference. 8 mLwere filled in a LiHep tube (lx, according to manufacturer's instruction and 4 rnL in a LiHep tube (2x concentration) and 1 mL in a LiHep tube (8x concentration) and the recovery to the reference serum was measured without LiHep. Recovery of cTnT values were within the acceptance criteria of + / - 10 % except for the highest concentration of LiHep (8x) when 20 mM / L spermidine was added to the assay formulation (Fig. 7 A, B). Higher concentrations of spermidine were also tested in the assay formulation (e.g. 30 and 40 mM / L) but higher concentrations led to loss of signal dynamic on counts but not on concentration level (not shown). Thus 20 mM / L spermidine is sufficient to reduce the LiHep interference significantly in the TnThs Gen6 assay formulation.

[0119] Example 4: Spermidine is efficient to reduce interference with LiHep for cTnlhs assay

[0120] It was tested whether LiHep can cause an interference with cardiac troponin I (cTnl). Using the same setup as above, simulating increasing LiHep concentrations by using serum with a defined concentration of either native (71 ng / L) or spiked rec. cTnl (74 ng / L). A negative influence of increasing amounts of LiHep on native cTnl was seen in the absence of spermidine in the reagent pack (Fig. 8 A). The addition of low concentrations already recover the samples so that they are within the acceptance criteria of + / - 10% and saw a recovery close to 100% with 18 mM / L, a similar concentration used in TnThs Gen6 (Fig. 8A). Increasing the concentrations showed better recovery but had a negative impact on counts for cTnl. In contrast to cardiac Troponin T (cTnT) as tested above, an influence of increasing amounts of LiHep was not see in the absence of spermidine when using the recombinant cTnl (rec. cTnl). Increasing the concentrations of spermidine increased the recovery but all within the acceptance criteria (Fig. 8B). The difference between native and rec. cTnl might be that the native cTnl in the blood of patients mainly exists as dimer of cTnl and troponin C (TnC) while the rec. cTnl is a construct only expressing cTnl without TnC (Wu, A. H. B. et al. Characterization of cardiac troponin subunit release into serum after acute myocardial infarction and comparison of assays for troponin T and I. Clin. Chem. 44, 1198-1208 (1998)). Thus, the circulating form of cTnl is very different from the rec. cTnl.

[0121] The influence on the recovery of different concentrations of native cTnl was tested in the absence of spermidine or increasing amounts of spermidine simulating lx concentration of LiHep (Fig. 9A) or 2x concentration of LiHep (Fig. 10B). The recovery in the absence of spermidine was out of the acceptance criteria of + / - 10% at 32.5 ng / L and 1345 ng / L cTnl and slightly within for the highest concentration of 4861 ng / L cTnl (Fig. 9A, B). The addition of spermidine of different concentrations efficiently removed the interference by LiHep and all tested samples were within the acceptance criteria.

[0122] In summary, Troponin I also has an overall negative charge, but the distribution of the negative charge is different than in cTnT and thus the mechanism of LiHep interference might be different than for cTnT. As seen by these experiments, there is a need to add spermidine to the assay formulation to account for possible LiHep interference in native samples.

[0123] The effect of spermidine on the recovery of cTnl in the presence of LiHep is not as pronounced as for cTnT and this might be the result of different distributions of charges across the cTnl molecule. The charge of cTnl alone and cTnl in complex with TnC was modelled. cTnl alone shows a clear positive charged pattern at the N-terminus in the spot where our Ru-labelled assay antibody 38A10 would bind and could thus interfere (not shown) with our assay. When the complex consisting of cTnl and TnC is modelled, the charges are almost equally distributed and no clear hotspot for binding of heparin is predicted. Since the literature in general assumes that Tnl is only present in complex with TnC the influence of heparin on native cTnl recovery is not solely explainable by the charge distribution of the complex. On the other hand one can assume that the literature only looks at high concentrations of cTnl in serum of patients, since the method to detect the different forms used, Size Exclusion Chromatography (SEC) is not sensitive enough to detect the different forms at low concentrations one could assume that free cTnl exists at low concentrations. Thus an interference elimination is needed for our new development of Tnlhs as well, which is supported by the data provided.

Claims

Claims1. A method for analyzing a protein analyte in a heparin-treated sample comprising or suspected to comprise said protein analyte, said method comprising the step of contacting said heparin-treated sample with spermidine prior to analyzing the said protein analyte in the said sample.

2. The method of claim 1, wherein said sample is a blood, serum or plasma sample.

3. The method of claim 1 or 2, wherein said analyzing of the protein analyte is carried out by using at least one binding agent which specifically binds to the said protein analyte selected from the group consisting of antibodies, aptamers, darpins, affibodies, and analyte binding proteins, such as protein or peptide receptors or ligands.

4. The method of any one of claims 1 to 3, wherein a protein analyte present in the heparin- treated sample is recovered by at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%.

5. The method of any one of claims 1 to 4, wherein said protein analyte is a positively- charged protein or peptide.

6. The method of claim 5, wherein said positively-charged protein or peptide is cardiovascular protein biomarker.

7. The method of claim 6, wherein said cardiovascular biomarker is selected from the group consisting of: a cardiac troponin, preferably troponin T or troponin I, a natriuretic peptide, preferably, NT-proBNP, pro BNP, BNP, or ANP, C-reactive protein (CRP), interleukin 6 (IL-6), an apolipoprotein and lipoprotein (a).

8. The method of claim 5, wherein said positively-charged protein or peptide is a neurological biomarker protein.

9. The method of claim 8, wherein said neurological biomarker is selected from the group consisting of Neurofilament (NFL), Tau, preferably, pTau217 or pTaul81, Neurotrophin (3 or 4), Myelin Basic Protein (MBP) or Synuclein.

10. The method of claim 5, wherein said positively-charged protein or peptide is a metabolic or cancer biomarker protein.

11. The method of claim 10, wherein said metabolic or cancer biomarker is selected from the group consisting of C reactive protein (CRP), IL-6, TNF-alpha or VEGF, an apolipoprotein, Transferrin, Ferritin, Insulin, Glucagon, PTH or Calcitonin.

12. A method for assessing a cardiovascular disease or disorder comprising the steps of: a) analyzing a protein analyte by the method of any one of claims 1 to 7; and b) assessing the cardiovascular disease or disorder based on the analyzed protein analyte.

13. A method for assessing a neurological disease or disorder comprising the steps of: a) analyzing a protein analyte by the method of any one of claims 1 to 5 or 8 or 9; and b) assessing the neurological disease or disorder based on the analyzed protein analyte.

14. A method for assessing a metabolic disease or disorder or a cancer comprising the steps of: a) analyzing a protein analyte by the method of any one of claims 1 to 5 or 10 or 11 ; and b) assessing the metabolic disease or disorder or a cancer based on the analyzed protein analyte.

15. Use of spermidine for analyzing a protein analyte in a heparin-treated sample comprising or suspected to comprise the protein analyte.

16. A kit for carrying out the method of any one of claims 1 to 14 comprising: i) spermidine; and ii) at least one binding agent which specifically binds to the said protein analyte selected from the group consisting of: antibodies, aptamers, darpins, affibodies, and analyte binding proteins, such as protein or peptide receptors or ligands.

Images