Patent Description:
Brain natriuretic peptide, also known as B-type natriuretic peptide, abbreviated BNP, is a hormone secreted by cardiomyocytes in the heart ventricles in response to stretching caused by increased ventricular blood volume, cardiac wall stress. NT-proBNP is produced by cleaving the proBNP into BNP and the NT-proBNP.

Measuring BNP and NT-proBNP is useful in the diagnosis and monitoring of heart failure. BNP and NT-proBNP cannot be compared directly, as BNP is the biological active metabolite and has a half-life (t<NUM>/<NUM>) of approx. <NUM> and does not degrade in the kidneys. It is only stable for a very short time in vitro.

NT-proBNP is however more stable, it is biologically inactive and filtered by the kidneys. Its half-life is about <NUM> to <NUM> hours and the concentration in the blood is higher than that of BNP. Compared to BNP, the half-life is highly dependent of kidney function. In vitro, NT-proBNP is more stable than BNP, and can be stored for at least three days at <NUM> - <NUM>. Consequently, proBNP and NT-proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure.

The recommended reference range limits to exclude heart failure in patients with non-acute symptoms are currently <NUM> ng/L and <NUM> ng/L for BNP and NT-proBNP respectively. The recommended reference range limits to exclude heart failure in patients with dyspnoea or increasing symptoms are higher, <NUM> ng/L and <NUM> ng/L for BNP and NT-proBNP respectively. The reference range is also wide, in particular for acute heart failure, and in particular for older patients, see Table <NUM>:.

NT-proBNP is a peptide chain of <NUM> amino acids with a mass of <NUM> Da in its non-glycosylated form. The amino acid sequence is shown in <FIG>. Before <NUM>, the glycosylation of proBNP was unknown. The first evidence of proBNP glycosylation was published in <NUM> (<NPL>). The presence of glycosylation and the observations of higher molecular weight isoforms of BNP have caused much confusion about the relevant forms of NT-ProBNP in the blood stream.

In <NUM>, it was shown that higher NT-proBNP concentrations were measured using the proBNP II assay on an Elecsys® platform (Roche Diagnostics) when the samples were pre-treated with a deglycosylation enzyme (<NPL>.

The exact glycosylation sites of endogenous proBNP and NT-proBNP were only recently characterized (<NPL>).

Sandwich immunoassays for the detection of NT-proBNP are commercially available, and disclosed for example in <CIT>) which relates to a sandwich assay using two antibodies which recognize different epitopes of the native NT-proBNP and at the same time can bind to said native NT-proBNP, wherein at least two antibodies bind in the region of amino acid <NUM>-<NUM> of N-terminal proBNP and the antibodies are obtained by immunizing a suitable organism with recombinant NT-proBNP.

<CIT>, <CIT> and <CIT>) define antibodies specific to epitopes on the proBNP molecule defined by amino acids no. <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>.

<CIT> and <CIT>. ) both relate to an immunoassay kit for detecting BNP, proBNP or a fragment thereof, in a sample, comprising: a first antibody or binding fragment thereof, specific for a region of a ring structure of BNP and proBNP; wherein the first antibody or binding fragment thereof, binds to BNP, proBNP or to a fragment thereof comprising the region thereof so as to form a first order immune complex; and a second antibody or binding fragment thereof, not recognizing free BNP, free proBNP or a free fragment thereof, or free first antibody, or which recognizes them with <NUM>-fold or less affinity than it recognizes the first order immune complex, wherein, the second antibody or binding fragment thereof, is Ab-BNP2 or Ab-BNP4.

<CIT>) relates to stable standards for BNP immunoassays and defines the use of a peptide selected from the group consisting of an isolated or recombinant or synthetic proBNP consisting of a specific amino acid sequence or a sequence that differs by five or fewer amino acid substitutions, insertions or deletions as a standard or calibrator in a method for detecting BNP immunoreactivity in a sample.

<CIT>) relates to a polyclonal-monoclonal ELISA assay for detecting N-terminal proBNP using isolated polyclonal antibodies specific for NT-proBNP within a certain range of amino acid residues, and isolated monoclonal antibodies specific for NT-proBNP within another, specific range of amino acid residues.

Comparison of existing clinical assays for the determination of NT-proBNP shows that the existing assays are still not standardised, and that the same samples yield different values depending on which assay is used. See for example <NPL>.

Nevertheless, NT-proBNP has been called "the gold standard biomarker in heart failure" (<NPL>). The current deviation between values obtained using different assays is however not satisfactory, and there is a need for an independent reference method for determining the concentration of NT-proBNP.

<CIT>) discusses the therapeutic use of proBNP in pharmaceutical compositions and determines high concentrations of recombinant proBNP in a regular buffer. Pollit et al. do not show, teach or suggest how physiological amounts of NT-proBNP could be determined in plasma samples.

<CIT>) discloses a method focused on degradation of proBNP and determining the presence or amount of one or more natriuretic peptides, or their fragments, in a sample, the method designed to distinguish between various natriuretic peptides.

In the article "<NPL>) note that there is no agreement on what kind of BNP or NT-proBNP standard should be used for calibration, and a certified reference material as well as reference measurement procedures are lacking.

<CIT> discloses a method for determining a concentration of human N-terminal pro-hormone BNP (NT-proBNP) in a sample but it does not use high affinity epitopes for a non-glycosylated epitope and there is no step of trypsinization.

The present inventors have addressed the above problem, and others, by making available a robust and reliable method suitable for serving either as a reference method for calibrating and ensuring the accuracy of existing methods, or as a novel method in clinical chemistry for accurately determining the concentration of NT-proBNP.

The broadest aspect of the claimed invention is defined in independent claim <NUM>. Preferred embodiments are defined in claims <NUM>-<NUM>.

The method defined in the claims qualifies as a higher order method or reference method, meaning that it is a high-accuracy method that can be used as a diagnostic method as such, but also for assigning values to calibration materials. Generally, the term reference method infers a high-quality method whose results are known to be correct. Any differences between a test method and a reference method are assigned to the test method, i.e., the errors are attributed to the test method because the correctness of the reference method is well documented.

According to a first aspect the present disclosure makes available a method for determining a concentration of human N-terminal pro-hormone BNP (NT-proBNP) in a sample, wherein said method comprises.

In the above sequences, (K/R) denotes trypsin cleavage sites.

According to an embodiment, freely combinable with any of the above embodiments, said at least one labelled peptide standard is full-length recombinant NT-proBNP.

According to an embodiment, freely combinable with any of the above embodiments, said at least one peptide standard is chosen from NHLQG(K) (SEQ ID NO. <NUM>), EVATEGI(R) (SEQ ID NO. <NUM>), and MVLYTL(R) (SEQ ID NO.

According to a specific embodiment, freely combinable with any of the above embodiments, said at least one peptide standard is EVATEGI(R) (SEQ ID NO. <NUM>), preferably a deuterium labelled EVATEGI(R) (SEQ ID NO.

According to an embodiment, freely combinable with any of the above embodiments, said affinity purification method makes use of affinity beads carrying at least one antibody against said NT-proBNP, and wherein said at least one antibody exhibits high affinity for a non-glycosylated epitope on the NTproBNP molecule. Preferably said affinity beads are magnetic affinity beads.

According to an embodiment, freely combinable with any of the above embodiments, said at least one antibody binds specifically to a non-glycosylated epitope on the NT-proBNP within an amino acid sequence chosen from SEQ ID NO. <NUM> and SEQ ID NO.

According to an embodiment of the first aspect, freely combinable with any of the above embodiments, said enzymatic digestion is a trypsinization. Preferably said trypsinization is performed while the labelled standard and native NT-proBNP are associated with the beads.

According to an embodiment, freely combinable with any of the above embodiments, quantitation is performed based on calibration against enzymatically digested recombinant NT-proBNP.

According to an embodiment, freely combinable with any of the above embodiments, the method is calibrated using trypsinated standard NT-proBNP at relevant concentrations, and wherein all standards are spiked with an internal standard. Preferably the internal standard is labelled recombinant full-length NT-proBNP.

Another aspect relates to a method for assigning a value to calibration materials, testing and/or quality control of assays for determining a concentration of human N-terminal pro-hormone BNP (NT-proBNP) in a sample, wherein said method comprises the steps outlined in the first aspect and embodiments thereof.

A third aspect of the present disclosure relates to a method for use in the diagnosis and monitoring of heart failure, wherein said method comprises the steps outlined in the first aspect and embodiments thereof.

Further aspects and embodiments and their advantages will emerge from the following detailed description, examples, and the attached claims and drawings.

Before the present invention is described in more detail, it is to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The term "sample" as in "a sample of whole blood" refers to a sample taken from a human or animal body, and which sample will not be returned to said human or animal body. There are standardized methods for obtaining and handling a blood sample taken from a human or animal body, involving the use of needles, syringes, micro cuvettes etc. These methods are well-known to persons skilled in the art. The currently most preferred type of sample is a lithium heparin treated sample of whole blood. There are several blood collection tubes containing spray coated lithium heparin readily available from various commercial supplies, e.g. the BD Vacutainer® available from BD, Oakville, Ontario, Canada.

The term "immunogen" in this disclosure refers to substances used to produce antibodies and includes conjugated and unconjugated forms.

The term "specifically binds to NT-proBNP within the amino acid sequence of. " is understood to mean that only amino acids within the specified amino acid stretch of NT-proBNP are responsible for being specifically recognized by and/or binding to the antibody. In other words, an antibody as described herein specifically recognizes and/or binds to an epitope within a given amino acid stretch of NT-proBNP.

The term "specifically recognizes an epitope" or "specifically binds an epitope" is understood to mean that only the amino acids of the epitope are responsible for the binding to the antibody. Accordingly, the term "epitope" denotes an antigenic determinant, namely a specific part or parts of an antigen such as NT-proBNP to which an antibody binds (such as an epitope comprising part or all amino acids found in the amino acid sequences corresponding to SEQ ID NO: <NUM> or <NUM>). None of the other amino acids of an antigen such as NT-pro-BNP are supposed to be involved in the binding of the antibody. Lack of cross-reactivity of an antibody to other molecules means that the antibody has high epitope specificity.

Further, "binding with substantially the same affinity" is understood to mean that an antibody binds for example to two or more different peptides with binding affinities in the same order of magnitude. The antibody or antibodies as described herein bind to a peptide, e.g. an <NUM>-mer peptide in the context of an epitope mapping assay, or to a protein, with an affinity of at least about <NUM>%, of at least about <NUM>%, of at least about <NUM>%, of at least about <NUM>%, of at least about <NUM>%, of at least about <NUM>%, of at least about <NUM>%, of at least about <NUM>%, or of at least about <NUM>% of the affinity of the same antibody or antibodies binding to another peptide or protein.

As used herein, the term "at least one antibody or antibodies" refers to polyclonal antibodies, a mixture of monoclonal antibodies of any isotype (IgA, IgG, IgD, IgM, IgY) or an antigen-binding fragment thereof, including but not limited to F(ab), F(ab'), F(ab')<NUM>, Fv fragments, single chain antibodies such as scFv, chimeric antibodies, humanized antibodies and a Fab expression library.

The terms "binding fragment of an antibody" or "antibody fragment" refer to a fragment of a full-length antibody which results e.g., from deletion of N-terminal or C-terminal amino acids of the full-length antibody and which maintains the capacity to bind the cognate antigen with about the same specificity and/or KD as the full-length antibody.

Assays to determine binding specificity and KD of an antibody are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see <NPL>.

One approach to measure specificity is by testing an antibody for binding to its cognate antigen and other non-related antigens using Western Blotting or ELISA. Another approach to measure affinity of binding of an antibody to its antigen is by surface plasmon resonance (SPR) using for example the Biacore™ assay platform and software (GE Healthcare).

Blood tests, involving laboratory examination of a sample of blood to obtain information about its physical and chemical properties, today form the foundation of medical diagnosis. Hundreds of haematological tests and procedures have been developed over the years. While the tests were originally performed manually, one by one, the field of clinical chemistry has become highly automated. Today multiple tests can be carried out simultaneously on one single sample of blood using automated clinical chemistry analysers, instruments frequently referred to as autoanalyzers.

While the presently disclosed assay method can be performed manually, the assay is preferably performed on an autoanalyzer, as this allows high throughput, reduces error, and minimizes staffing requirements. A closed, automated sample handling also minimizes the risk that staff is exposed to possible blood borne pathogens.

When taking a blood sample, blood is usually drawn from a vein in the arm and collected in standardized sterile blood collection tubes. Different tubes are available, depending on which component or components that is to be analysed. A blood collection tube can be empty, or prefilled with a buffer or more frequently, with an anticoagulant. Examples of anticoagulants include sodium citrate, lithium-heparin, and EDTA. Different combinations are also possible, there are for example tubes containing sodium heparin and EDTA. The Vacutainer™ (BD, Becton, Dickinson and Company) is one example of blood collection tubes, available in different sizes and pre-filled with various reagents.

A distinction is made between plasma samples and serum samples. When a plasma sample is desired, blood is collected in a tube containing an anticoagulant and the tube is turned upside down about <NUM> to <NUM> times directly after filling, in order to ensure mixing. The tubes are then stored in an upright position in room temperature to avoid haemolysis. EDTA and lithium heparin containing tubes can generally be stored up to <NUM> hours in room temperature. Alternatively, the tubes can be deep-frozen and stored for later analysis.

For obtaining a serum sample, blood is collected in an empty sample tube or a tube containing a coagulation activator. After filling, the tubes are turned upside down about <NUM> to <NUM> times directly after filling, in order to ensure mixing. The tubes are then stored in an upright position in room temperature to avoid haemolysis. In most cases, serum tubes can be stored in room temperature and without centrifuging for up to <NUM> hours. Upon arrival in the laboratory, the tubes are centrifuged, for example centrifuged for <NUM> minutes at <NUM> x g. Following centrifugation, a layer of blood cells can be seen at the bottom of the tube, and the fluid above is called serum. The serum should be separated and stored cold without any additives until transport or analysis.

The inventors note that immunoassay systems require calibration protocols that are normally more sophisticated than many analytical techniques in routine clinical use. Calibrators used in such assays may differ significantly from the analyte in clinical specimens. Differences in the properties of calibrators, or reference materials, from those of clinical specimens may include the species origin of the calibrator for an analyte; integrity of the molecular species; matrix of the calibration solution, and addition of preservative agents.

Brain natriuretic peptide (BNP) and the N-terminal fragment of the BNP precursor (NT-proBNP) are widely used biomarkers for heart failure (HF). Since the discovery of BNP in <NUM>, much effort has been allocated to the precise detection of BNP and NT-proBNP levels for reliable HF diagnostics. As a result, measurements of these biomarkers are globally accepted and used in clinical practice for the diagnosis of acute and chronic HF, risk stratification, and monitoring response to therapy. Several immunoassays specific for BNP and NT-proBNP are currently commercially available.

The multiple circulating BNP fragments, along with proBNP and NT-proBNP, collectively form the B-type natriuretic peptide family. Immunoassays for BNP utilize a variety of antibodies, both monoclonal and polyclonal, and diverse calibrator materials. Therefore, there are substantial differences in patient test values between BNP methods, even for assays that use identical antibody configurations on different analytical platforms.

Previous studies have demonstrated that proBNP and NT-proBNP are glycosylated to varying degrees that can interfere with commercial immunoassays used to quantify NPs in clinical practice (e.g., Saenger et al. The extent to which these immunoassays exhibit cross-reactivity to B-type natriuretic peptides as well as with their glycosylated and non-glycosylated forms is an important question to elucidate, as this may affect the clinical performance of the assays with implications for patient care. Furthermore, there are currently no formal efforts to standardize or maintain harmonization of BNP or NT-proBNP assays, primarily owing to the known differences in antibodies used and lack of a primary reference standard material.

Recent comparative studies however indicate that there are marked differences between different BNP and NT-proBNP assays and platforms, and the results of measurements are not sufficiently comparable. One such comparison is presented in Example <NUM> (Collin-Chavagnac et al. The present inventors realized the significance and consequences of this, and the method outlined in the present description and claims offers an advantageous solution.

This lack of equivalence between the assays complicates the interpretation of the results and as a consequence, the reference range limits used for diagnostic decisions are in fact method dependent. As a consequence, diagnosis and decisions regarding therapy may vary, or even be arbitrary depending on which method was used for analysing the patient's samples. Presently, there also does not appear to be any agreement on what kind of BNP or NT-proBNP standard should be used for calibration, and certified reference material as well as reference measurement procedures are lacking.

According to another embodiment, freely combinable with any of the above embodiments, one peptide standard is used quantitatively, to confirm the quantification of NT-proBNP in the sample, whereas another peptide standard is used qualitatively, to confirm that it actually is NT-proBNP that is detected and quantified in the assay.

According to an embodiment, freely combinable with any of the above embodiments, said affinity purification method makes use of affinity beads carrying at least one antibody against said NT-proBNP, and wherein said at least one antibody exhibits high affinity for a non-glycosylated epitope on the NTproBNP molecule. Preferably said affinity beads are magnetic affinity beads. Said at least one antibody is chosen from monoclonal and polyclonal antibodies exhibiting high affinity for a non-glycosylated epitope on the NTproBNP molecule.

In the past, determining the true concentration of NT-proBNP has been challenging, as the variations between results obtained with different assays has shown. The approach outlined above has several advantages, one being that variations in glycosylation no longer impact the results. Another advantage is that the herein disclosed method has a significantly improved sensitivity. The inventors have also proven that the method is robust and transferrable to different sites, where it has been performed by different operators and on different instruments. The claimed method can thus function as an important tool in research, in the calibration of existing assays, for the development of new assays, and as a diagnostic assay in itself.

The claimed method thus presents an independent approach to quantifying NT-proBNP and offers a possibility to calibrate and standardize existing methods, and it will be useful in the production of standards, quality assurance and in the supervision of clinical laboratories. In other words, the invention offers an approach to level up existing assays.

Further, the claimed method exhibits a significantly improved sensitivity, making it possible to detect and accurately quantify a low-abundant protein such as NT-proBNP. The claimed method can also be used to generate data making it possible to recalculate and transfer results obtained with different methods, thus creating an uninterrupted medical history for a patient who has been subject to different analyses during the course of his/her illness, and in contact with various care providers.

Above all, the present method makes available a reliable method for detecting and quantifying an elusive biomarker.

Specific advantages have been presented in association with selected features and other advantages will be apparent to a person skilled in the art upon study of the following examples which illustrate the inventions.

In the article "<NPL>et al. report the results of their comparison of NT-proBNP and BNP levels in fresh samples from heart failure (HF) patients measured using <NUM> different immunoassays. NT-proBNP (CobasH232®, Elecsys®, Vidas®, Vista®, XPand®, Vitros®) and BNP (Triage®, Access®, CentaurXP®, Architect®) levels were measured in <NUM> heparin and <NUM> EDTA samples, respectively.

The Pearson correlation coefficient ranged between <NUM> (Triage®-Centaur®) and <NUM> (Access®-Architect®) for BNP assays and between <NUM> (Vidas®-Cobas H232®) and <NUM> (Vitros®-Vidas®) for NT-proBNP assays. Passing Bablok regression analyses showed a significant difference in the slopes [<NUM> (Centaur®-Triage®) to <NUM> (Architect®-Centaur®)] and intercepts [-<NUM> ng/L (Architect®-Centaur®) to <NUM> ng/L (Access®-Triage®)] for BNP assays, and a lower heterogeneity between NT-proBNP assays [<NUM> (Vidas®-Elecsys®) to <NUM> (Vitros®-Vidas®) and -<NUM> ng/L (XPand®-CobasH232®) to <NUM> ng/L (CobasH232®-Elecsys®) for slopes and intercepts, respectively].

The concordance correlation coefficient revealed a poor (pc<<NUM>) to moderate (pc=<NUM>-<NUM>) agreement in <NUM>/<NUM> pairs of BNP assays and an almost perfect (pc><NUM>) agreement in <NUM>/<NUM> pairs of NT-proBNP assays. The acceptable difference limit reflecting the number of individual discrepant results between two assays, ranged between <NUM>% (Access®-CentaurXP®) and <NUM>% (Architect®-Triage®) for BNP assays, and between <NUM>% (Vidas®-Vitros®) and <NUM>% (CobasH232®-Xpand®) for NT-proBNP assays.

Collin-Chavagnac et al. concluded that their study stresses the lack of transferability of the results obtained using different techniques to measure BNP and NT-proBNP levels in fresh samples, adding that "individual reference ranges and HF diagnostic cut-offs should be assessed for each commercial NP immunoassay". The authors recommended that HF patients should be systematically monitored using the same assay (BNP or NT-proBNP) over the time (Collin-Chavagnac et al.

Antibodies were produced in either a mammalian host (rabbit or goat), or in an avian host (hens) against different immunogens and purified as shown in Table <NUM> below.

Antibodies against NT-proBNP and variants thereof (for example recombinant or native, truncated or non-truncated forms, glycosylated and non-glycosylated forms, and combinations thereof) can be produced according to methods known to a person skilled in the art, either in mammal hosts, e.g., goats or rabbits, or preferably in hens, in which case the antibodies can be isolated from the egg yolk. Two peptides designated "Peptide <NUM>" and "Peptide <NUM>" were synthetized (Genscript Biotech (Netherlands) B. , Leiden, The Netherlands), delivered as a white lyophilized powder with > <NUM> % HPLC purity, and used as immunogens, in addition to the recombinant, full-length NT-proBNP in glycosylated and non-glycosylated form:.

When raising antibodies against a shorter peptide, such as Peptide <NUM> or Peptide <NUM>, these are preferably conjugated to a suitable carrier to increase immunogenicity. One example is keyhole limpet hemocyanin (KLH) used in the immunization of the mammalian hosts (rabbits). Immunization and antibody purification protocols for raising polyclonal rabbit antibodies are well-known to a person skilled in the art, and available for example from CBG, Max Planck Institute of Molecular Cell Biology and Genetics. General procedures are also disclosed in <NPL>.

Avian immunoglobulin fractions such as those used in this assay can be purified from eggs from immunized hens, immunized with native or recombinant NT-proBNP or variants thereof, as indicated in Table <NUM>. The antigen and suitable adjuvant, e.g., complete Freund's adjuvant, is injected subcutaneously and/or intramuscularly into breast tissue of the hen. Immunizations are repeated at regular intervals, for example on day <NUM>, <NUM> and <NUM>. Antibodies are usually detected in the eggs by day <NUM>.

Eggs are collected, the yolk separated from the white, and the immunoglobulin fraction is isolated from the egg yolk. As a first step, the lipids and lipoproteins are removed, using a suitable method known to persons skilled in the art, for example precipitation using PEG or dextran sulphate, solubilisation using organic solvents, or ultrafiltration. The resulting substantially lipid-free solution can then be concentrated and purified to obtain the desired immunoglobulin fraction containing polyclonal antibodies against the antigen.

The immunisation of rabbits was performed by GenScript Biotech Corp. , Piscataway, NJ, USA, and the immunization of hens by Norwegian Antibodies AS, NABAS, Ås, Norway. All antibodies were purified by Getica AB, Gothenburg, Sweden.

Goat IgG was produced by InnovaGen AB, Lund, Sweden, immunizing goats with a peptide according to SEQ ID NO. <NUM> and SEQ ID NO. <NUM>, following the same methods, mutatis mutandis. Methods for the concentration and/or purification of immunoglobulins are also well known to a person skilled in the art and include precipitation steps using for example PEG or sodium sulphate, or other well-known methods such as ultrafiltration or liquid chromatography. For more guidance, see e.g.<NPL>.

Polyclonal antibodies against the peptides of SEQ ID NO <NUM> and SEQ ID NO. <NUM> also achieved high affinity (See Example <NUM> below) and high specificity, avoiding binding to glycosylated regions, which allows the measurement of the true NT-proBNP concentration with high sensitivity also in a turbidimetric or nephelometric setting.

As part of the quality assurance in the production of antibodies for the experiments disclosed herein, affinity analysis was routinely performed for each batch. The affinity of an antibody for its antigen is determined by surface plasmon resonance (SPR) in a method often referred to as the Biacore™ method. SPR occurs under conditions of total internal reflection of plane-polarized light at an interface between two media of different refractive index, coated with an electrically conducting film. When a sample binds to the surface, the refractive index increases, and the resonance angle changes. The angle is monitored continuously as the SPR signal, and a plot of the signal against time, called a sensorgram, shows the changes in refractive index as a sample binds to and dissociates from the surface.

The analysis was performed using a Biacore X100 system, using the GE Sensor Chip CM5 (all from GE Healthcare). The chip has a gold surface with a dextran matrix having carboxyl groups on it. For determining the affinity of an antibody to an antigen, the surface of a chip is activated, and an antigen is covalently attached to the surface. This process is irreversible, so the chip can be re-used several times without the need for extra antigen.

For determining the kinetic constants, the target antibodies were first dialysed against PBS and then diluted in HBS to <NUM> nanomolar (nM). A dilution series was prepared (<NUM> - <NUM> - <NUM> - <NUM> and <NUM>) for each target antibody. The concentration was confirmed by spectrophotometric analysis at <NUM>.

The antibodies were analysed on the Biacore X100 system, using the Biacore X100 Control Software, which when executed on the system injects the antibody dilution series in reverse (starting with the most diluted) sequentially and as a final step the surface is re-generated with glycine (pH <NUM>). The kinetic constants are extracted from the sensorgram using the Biacore X100 Evaluation Software based on a <NUM>:<NUM> kinetic model. Typical results of the Biacore™ analysis are summarized in Table <NUM> below:.

The results showed that the immunisation protocol and purification consistently produced antibodies binding specifically to NT-proBNP and exhibiting a KD to recombinant NT-proBNP of less than <NUM>.

The affinity constants were plotted against the span response and the sensitivity of the assay when run on a clinical chemistry analyser (Mindray BS-<NUM>). This showed a clear correlation between span, sensitivity, and the dissociation constants.

IgG antibody against the peptide of SEQ ID NO <NUM> from goat antiserum, purified against the same peptide, was tested against a Biacore™ chip with the peptide of of SEQ ID NO <NUM>. The equilibrium dissociation constant was determined to KD= <NUM>.

IgG antibody against the peptide of SEQ ID NO <NUM> from goat antiserum, purified against the same peptide, was tested against a Biacore™ chip with the peptide of of SEQ ID NO <NUM>. The equilibrium dissociation constant was determined to KD= <NUM>.

Similarly, IgG antibody against the peptide of SEQ ID NO <NUM> from rabbit antiserum was tested against a Biacore™ chip with the peptide of of SEQ ID NO <NUM>. The equilibrium dissociation constant was determined to KD= <NUM>. 54E-<NUM> and <NUM>.

Epitope mapping was performed by Pepscan Presto BV, Lelystad, The Netherlands, using libraries of linear and looped peptide arrays synthesised onto a solid support covered with a hydrogel. The mapping technique was first published in <NUM> (<NPL>). Chicken and rabbit antibodies were provided by Gentian AS, and the mapping focused on a <NUM> residues long segment of the NT-proBNP sequence, positions <NUM> - <NUM>, sequence SASDLETSGLQEQRNHLQGKLSELQV (identical to SEQ ID NO.

Synthesis of peptides: To reconstruct epitopes of the target molecule a library of peptide-based peptide mimics was synthesized using Fmoc-based solid-phase peptide synthesis. An amino functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxy-benzotriazole (HOBt) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA). Standard Fmoc-peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS liquid handling stations (<NPL>; and <NPL>).

In a first set, linear peptides of a length ranging from <NUM> - <NUM> derived from the target sequence /SEQ ID NO. <NUM>) were synthetized with an offset of one residue. In a second set, constrained peptides of length <NUM> - <NUM> were synthetized starting from Linear peptides of length <NUM>-<NUM> residues derived from the target sequence of NT Pro BNP with an offset of one residue were placed between two Cys residues. Cys residues were joined by mP2 CLIPS in order to create a loop mimic.

The binding of antibody to each of the synthesized peptides was tested in an ELISA. The peptide arrays were incubated with primary antibody solution overnight at <NUM>. After washing, the peptide arrays were incubated with a <NUM>/<NUM> dilution of an appropriate antibody peroxidase conjugate for one hour at <NUM>. After washing, the peroxidase substrate <NUM>,<NUM>'-azino-di-<NUM>-ethylbenzthiazoline sulfonate (ABTS) and <NUM>µl/ml <NUM> % H<NUM>O<NUM> were added. After one hour, the color development was measured. The color development was quantified with a charged coupled device (CCD) -camera and an image processing system.

The main epitope candidates identified in this study are presented in the table below, as well as in the enclosed sequence listing:.

The epitope mapping confirmed that the antibodies bind to the correct portions of the NT-proBNP molecule.

Superparamagnetic beads with a diameter of <NUM> containing surface epoxy groups were chosen for this example. <NUM> Dynabeads™ M-<NUM> Epoxy were weighed out in a tube. <NUM>µL distilled water was added. The mixture was vortexed for <NUM> sec and sonicated with a sonication probe for <NUM> seconds. Supernatant was removed by placing the tube on a magnetic rack. This was repeated once.

Goat anti-NT-proBNP antibodies and rabbit anti-NT-proBNP antibodies (produced as shown in Example <NUM>) were coated, separately, onto the Dynabeads®. The same coating procedure was used for both batches. The antibodies were buffer exchanged into <NUM> sodium phosphate buffer, pH <NUM> before coating. <NUM>µg antibody in a total of <NUM>µL <NUM> sodium phosphate buffer, pH <NUM> was reacted to the beads. The beads were vortexed for <NUM> sec before <NUM>µL <NUM> ammonium sulphate [(NH<NUM>)<NUM>SO<NUM>] in <NUM> sodium phosphate buffer pH <NUM> was added. The mixture was vortexed for <NUM> sec and incubated with mixing at <NUM> overnight.

The particle mixture was mixed on a roller mixer for <NUM> hours in room temperature, before the supernatant was removed, and the beads were washed in <NUM>µL <NUM> glycine pH <NUM> buffer, vortexed for <NUM> sec, separated on a magnet (<NUM>) and supernatant removed. The beads were then washed in <NUM>µL <NUM> Glycine pH <NUM>, and the same procedure followed. Then <NUM>µL <NUM> mMTris-HCl buffer, <NUM> NaCl pH <NUM> was added to the beads, the beads were placed on a roller mixer for <NUM>, separated on a magnet (<NUM>) and supernatant removed. Finally, <NUM>µL <NUM> Tris-HCl buffer, <NUM> NaCl pH <NUM> was added to the beads, the beads were placed on a mixer and incubated at <NUM> overnight. The following morning, the beads were put on a roller mixer for <NUM> hours, separated on a magnet (<NUM>) and supernatant removed. The Dynabeads® were suspended in <NUM> Tris-HCl buffer, <NUM> NaCl pH <NUM>, to a final concentration of <NUM>/mL.

The calibrator matrix can be constituted by serum or plasma materials from human or animal serum or plasma materials, or a buffer solution, comprising a known amount of the antigen, which can be human, recombinant, or synthetic NT-proBNP, or fragments thereof, such as peptide <NUM> (SEQ ID NO <NUM>), Peptide <NUM> (SEQ ID NO <NUM>), optionally conjugated to a carrier protein. The calibrator material can further comprise a salt solution, soluble polymers such as PEG-<NUM> (or other PEG materials) and preservative agents.

The composition of the calibrator can be adjusted until the assay method generates results corresponding to values obtained using a reference method as described herein, where the concentration of the calibrator is assigned by an immunoassay-based method using an antibody which binds specifically to a non-glycosylated epitope on the NT-proBNP molecule.

The concentration assignment of the calibrator material is well described in the prior art, for example in <NPL>.

Recombinant NT proBNP (8NT2, HyTest) was trypsinated in solution; <NUM>µl of NT proBNP standard in <NUM> ammonium bicarbonate was denatured with <NUM>µl Trifluoroetanol (TFE), reduced with <NUM>µl <NUM> dithiothreitol (DTT) at <NUM> for <NUM>; and alkylated with <NUM>µl <NUM> iodoacetamide (IAA) at room temperature in the dark for <NUM>. Thereafter <NUM>µl <NUM> DTT was added to remove excess IAA, then the mixture was kept in room temperature in the dark for <NUM>.

TFE concentration was diluted <NUM>-fold with <NUM> ammonium bicarbonate. Trypsin (sequencing grade, Promega) was diluted in ammonium bicarbonate and added at a protein to enzyme ratio of <NUM>:<NUM>. Digestion was performed overnight at <NUM>, stopped by addition of <NUM>µl formic acid after which the resulting peptides were desalted on C18 SPE columns.

Isotope labelled (deuterium labelled) peptide standards were ordered from ThermoFisher following instructions from the inventors. Heavy peptide NHLQG(K) (SEQ ID NO. <NUM>) contained labelled Lysine (+<NUM> Da), heavy peptide EVATEGI(R) (SEQ ID NO. <NUM>) contained labelled Arginine (+<NUM> Da), and heavy peptide MVLYTL(R) (SEQ ID NO. <NUM>) contained labelled Arginine (+<NUM> Da). Alternatively, heavy-labelled (15N) full-length NTproBNP from Promise Proteomics, Grenoble Cedex, France, was used for calibration and sample control.

The desalted peptides and labelled peptide standards were dried to dryness via vacuum centrifugation and stored frozen.

A plasma or a serum sample is a highly complex environment, containing multiple biologically active components, and additionally, as can be seen in <FIG>, BNP has many molecular forms. The identification of three specific peptide standards makes it possible to use one as the main standard for quantification, and one or two for a secondary, qualitative confirmation that it actually is NT-proBNP that is detected and quantified.

Sample preparation was performed by affinity purification of NT-proBNP from plasma/serum samples using magnetic affinity beads (Dynabeads®), prepared as described above in Example <NUM>. The beads were washed in PBS prior to use. Both rabbit and goat antibody carrying beads were used (separately).

Plasma samples were processed as follows; plasma (<NUM>) was mixed with <NUM>µl Dynabeads®, placed on mixer and incubated over-night at <NUM>. Following incubation, the beads were placed in a magnetic rack and the supernatant was removed. The beads were thereafter washed in PBS (V = <NUM> x <NUM>) and thereafter washed in <NUM> <NUM> ammonium bicarbonate.

Trypsination was performed on-bead in <NUM>µl <NUM> ammonium bicarbonate following a previously described protocol [<NPL>] with some modifications; NT-proBNP attached to affinity beads (<NUM>µl of beads in ammonium carbonate) was reduced with <NUM>µl <NUM> dithiothreitol (DTT) at <NUM>, <NUM> rpm for <NUM>; and alkylated with <NUM>µl <NUM> iodoacetamide (IAA) at room temperature in the dark for <NUM>. Thereafter <NUM>µl <NUM> DTT was added to remove excess IAA. Trypsin (sequencing grade, Promega) was diluted in ammonium bicarbonate and added in excess, at a ratio guaranteeing that all protein is degraded.

Digestion was performed overnight at <NUM>. The tubes with magnetic beads were placed in the magnetic rack and the tryptic peptide solutions were transferred to new tubes and centrifuged at <NUM> rpm for <NUM> to sediment any remaining trace particles. Tryptic peptide solutions were transferred to new tubes and <NUM>µl formic acid was added. The resulting peptides were desalted on C18 SPE columns.

The resulting peptides (from both standards and affinity samples) prepared as shown in Example <NUM>, were then desalted on C18 spin columns (C <NUM> Spin Columns (Pierce, Rockford, USA)). The C18 spin columns were then washed with 2x200 µL <NUM> % ACN and conditioned with 2x200 µL <NUM> % TFA in <NUM> % ACN prior to use. After sample (<<NUM> ug total protein) was added to the spin columns, they were washed with 2x200 µL <NUM> % TFA in <NUM> % ACN to remove salt. Peptides were eluted in two portions of <NUM>µL <NUM> % ACN with <NUM> % FA. Eluates were dried to dryness via vacuum centrifugation and stored frozen until reconstitution and analysis.

Alternatively, the peptides from both standards and samples were desalted on C18 Bond Elut LRC-C18 SPE columns (Agilent Technologies Inc. The columns were washed with <NUM> <NUM> % ACN and conditioned with <NUM> <NUM> % ACN with <NUM> % TFA prior to use. After sample (<NUM>µl trypsinated peptides plus <NUM> H2O) was added to the spin columns, they were washed with <NUM> <NUM> % ACN with <NUM> % TFA to remove salt and allowed to dry for <NUM> to <NUM> minutes. Peptides were eluted either in four portions of <NUM> or two portions of <NUM> <NUM> % ACN with <NUM>,<NUM> % FA. Eluates were dried to dryness via vacuum centrifugation and stored frozen until reconstitution and analysis.

For an overview of immunocapture bottom-up liquid chromatography mass spectrometry, see e.g. <NPL>, and <NPL>.

This analysis method included the following three peptides: NHLQG(K) (SEQ ID NO. <NUM>), EVATEGI(R) (SEQ ID NO. <NUM>), and MVLYTL(R) (SEQ ID NO.

The corresponding heavy peptides were used for quantitation. The signals for oxidized peptides were also monitored in the method.

<NUM> samples for LC-MS/MS analysis were reconstituted in <NUM>µl of <NUM>% ACN with the addition of heavy peptides. The heavy peptides were added to concentrations of <NUM> pmol/µl for EVATEGIR/MVLYTR (SEQ ID NO:s <NUM> and <NUM>) and <NUM>,<NUM> pmol/µl NHLQGK (SEQ ID NO <NUM>).

When using 15N full length NT-proBNP, the standard was added to the plasma prior to the affinity purification on beads and trypsinated together with the samples.

The LC-MS analyses were performed on an Agilent <NUM> Series LC/MS system (Agilent Technologies Inc. , Palo Alto, CA, USA). The system comprised a G4220A binary pump, a G4226A autosampler, a G1316C column thermostat and a G6490 triple quadrupole mass spectrometer.

The LC-MS analysis was performed in reversed phase mode employing an Ascentis Express Phenyl Hexyl (<NUM> x <NUM>, <NUM>) and gradient elution using <NUM> formic acid as eluent A and acetonitrile as eluent B. The gradient started at <NUM>% eluent B for <NUM> after which percentage B was increased to <NUM>% over <NUM> minutes, held constant at <NUM> % for <NUM> minute and thereafter returned to the starting conditions for system equilibration. The flowrate was <NUM>/min and the column temperature was maintained at <NUM>. All analyses were performed with electro spray ionization (ESI) in positive mode. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode. See Table <NUM> for the MRM-settings used.

Abbreviations: CE = collision energy, Cell acc = cell acceleration voltage. Instrument settings: Fragmentor <NUM> V. Gas Temp (°C): <NUM>, Gas Flow (I/min): <NUM>, Nebulizer (psi): <NUM>, Sheath Gas Temp (°C): <NUM>, Sheath Gas Flow (l/min): <NUM>, Capillary (V) <NUM>, Nozzle Voltage/Charging (V): <NUM>.

*Oxidised peptides. Monitored to assess oxidation during sample work-up. No specific standards available.

Three tryptic peptides were monitored, NHLQG(K), EVATEGI(R) and MVLYTL(R) (SEQ ID Nos <NUM>, <NUM>, and <NUM> respectively) along with the corresponding heavy-peptides (heavy-N labelled) originating from the internal standard N15-labelled NT-proBNP. For peptide MVLYTL (R), MRM transitions for an oxidised variant of the peptide and corresponding oxidised version of the heavy-labelled peptide were also monitored in the method. Multiple transitions were monitored for each peptide with the most abundant being used for quantitation and additional transitions used for qualification. Trypsinated standards of NT-pro-BNP at <NUM>-<NUM> ng/ml were used in calibration.

Data analysis was performed using Agilent MassHunter Qualitative and Quantitative analysis software. Quantitation was based on calibration against trypsinated standard NT-proBNP spike in in plasma at <NUM>-<NUM>-<NUM>-<NUM>-<NUM>-<NUM> ng/ml. Sample quantitation was also performed based on standard addition results. All standards and samples were spiked with <NUM> ng/ml of internal standard (15N-labelled NTproBNP). Three peptides were monitored (NHLQG(K), EVATEGI(R) and MVLYTL(R)). Heavy peptides (15N-labelled from 15N-labelled NTproBNP) were used as internal standards. Peptides EVATEGI(R) and MVLYTL (R) were shown to have the best response. They exhibited LOD/LOQ corresponding to <NUM> and <NUM> ng/ml NT-ProBNP and linear response to <NUM> ng/ml. Quantitation was done based on the EVATEGI(R) peptide (SEQ ID NO. Multiple transitions were monitored for each peptide with the most abundant being used for quantitation and additional transitions used for qualification, i.e., the verification that the correct/selected peptide is measured (and not an unrelated peptide with the same m/z). Here, peptide NHLQG(K) (SEQ ID NO. <NUM>) was only used for qualification.

The results from evaluating a pool of <NUM> samples are presented in Table <NUM> below, where the results obtained with a prior art method are compared to the results obtained with the method disclosed in the present application. The last column shows the ratio between the results, indicating that the disclosed in the present application consistently result in a higher reading, and that the difference is more pronounced at lower NT-proBNP concentrations.

Within the clinically relevant range, <NUM> - <NUM> ng/L as determined by a prior art method (the Roche Elecsys® NT-proBNP assay), the results exhibit a linear correlation (R<NUM> = <NUM>) as shown in <FIG>. The graph shows that the commercial assay consistently underestimated the concentration of NT-proBNP in the mentioned concentration range and that the comparison exhibits a linear correlation (R2=<NUM>).

For all <NUM> samples, in an interval of <NUM> - <NUM> ng/L as determined by the prior art method, the correlation is still very good (R<NUM> = <NUM>).

For three samples, the stability of the method was tested by performing analysis of the same samples on two consecutive days. The results are presented in Table <NUM>:.

As can be seen from the results, the method was reproducible and stable in the clinically relevant range.

During the priority year, a larger number of samples covering a wider concentration range (<NUM> to <NUM> ng/L) were investigated, and a comparison made between the results obtained with the claimed method and a commercial assay, the Roche Elecsys® NT-proBNP assay, performed according to the manufacturer's instructions. The results are shown in <FIG>. This graph confirms the results presented in <FIG> but also reveals that the concentration measurements between the commercial assay and the herein presented method converge in the higher concentrations. Both methods exhibit a good (polynomial) fit over a large concentration range (R<NUM> = <NUM>).

To investigate the utility of the inventive method, both plasma and serum samples were analysed using on the one hand the Siemens ADVIA Centaur® Immunoassay System (for plasma samples) and on the other hand the Siemens IMMULITE Immunoassay System (for serum samples). Both assays were performed according to the manufacturer's instructions. The results were compared to the concentrations determined using the herein claimed LC-MS/MS method.

<FIG> shows that the Siemens ADVIA assay underestimated the concentration of NT-proBNP in the lower concentration range, but that the comparison with concentrations determined using the claimed method exhibits a linear correlation (R2=<NUM>).

<FIG> shows the result for serum samples using another commercial method, the Siemens IMMULITE. The graph confirms the results presented in <FIG>, showing again an underestimation of the NT-proBNP concentration in the lower concentration range by the commercial assay, but also a convergence of results between the commercial and the herein presented method in the higher concentrations for different sample types. Both methods exhibit good (polynomial) fit over a larger concentration range (R2 = <NUM>).

The method disclosed herein was set up at two geographically different sites here denoted "<NUM>" and "<NUM>" and two operators at each site were trained to perform the analysis. At the first site, the measurements were made using a LC-ESI-Triple Quadrupole instrument (Agilent, USA) which is a liquid chromatography - electrospray ionization tandem mass spectrometer consisting of two quadrupole mass analysers in series, with a (non-mass-resolving) radio frequency-only quadrupole between them to act as a cell for collision-induced dissociation. At the second site, a nano-LC-ESI-Orbitrap instrument (Thermo Fisher Scientific, USA) was used.

In an initial comparison, <NUM> plasma samples with distinct concentrations (determined using the Roche method) were measured at both sites, and the measurements were made independently by two operators at each site. The operators were denoted A, B, C and D. The results are shown in Table <NUM>.

Overall, the results indicate that the herein disclosed assay is robust, stable, and capable of detecting the total amount of NT-proBNP in a sample, i.e. that the method detects NT-proBNP independent of its degree of glycosylation.

The inventors have now shown, based on the isolation and quantification of NT-proBNP using different immunoassay-based reference methods, involving the use of antibodies specifically binding to non-glycosylated epitopes of NT-proBNP both for determining the NT-proBNP concentration in the calibrator sample and in the test or patient sample, that a significantly higher concentration of NT-proBNP is detected in the test or patient sample. This is held to be the "true" concentration of NT-proBNP, independent of degree of glycosylation and other variables. Thus, a more reliable, and glycosylation-independent assay principle has been developed and its utility and robustness has been confirmed.

The method disclosed herein can be used to calibrate / validate existing assays or used as a stand-alone method in clinical chemistry. This will enable a more reliable and correct diagnosis of heart failure, in particular congestive heart failure. Further, as it has been shown that this approach is applicable to turbidimetry and nephelometry, this opens up the possibility of automated, cost-efficient and yet reliable methods for diagnosing of heart failure or an elevated risk for heart failure, monitoring a subject with heart failure or having an elevated risk for heart failure, or assessing the severity of heart failure, by determining the concentration of NT-proBNP in a bodily fluid sample of said subject suspected of having heart failure.

Without further elaboration, it is believed that a person skilled in the art can, using the present description, including the examples, utilize the present invention to its fullest extent. Also, although the invention has been described herein with regard to its preferred embodiments, which constitute the best mode which is set forth in the claims appended hereto.

Claim 1:
A method for determining a concentration of human N-terminal pro-hormone BNP (NT-proBNP) in a sample, characterized in that the method comprises
- extracting and isolating NT-proBNP from the sample using an affinity purification method making use of affinity beads carrying at least one antibody against said NT-proBNP, and wherein said at least one antibody exhibits high affinity for a non-glycosylated epitope on the NTproBNP molecule,
- adding a least one labelled peptide standard to said isolated NT-proBNP,
- subjecting said isolated NT-proBNP and peptide standard to enzymatic digestion wherein said enzymatic digestion is a trypsinization, and wherein said trypsinization is performed while the labelled standard and native NT-proBNP are associated with said affinity beads, producing a mixture of peptides, and
- detecting at least one peptide in the resulting mixture, using liquid chromatography-mass spectrometry analysis with subsequent mass spectrometric detection, wherein said at least one peptide to be detected is chosen from NHLQG(K) (SEQ ID NO. <NUM>), EVATEGI(R) (SEQ ID NO. <NUM>), and MVLYTL(R) (SEQ ID NO. <NUM>).