NO348199B1

NO348199B1 - Immunoassay and method for determining secretoneurin peptide concentration

Info

Publication number: NO348199B1
Application number: NO20230790A
Authority: NO
Inventors: Frank Frantzen; Arne Ludvig Faaren
Original assignee: CardiNor AS
Priority date: 2023-07-13
Filing date: 2023-07-13
Publication date: 2024-09-30
Also published as: NO20230790A1; WO2025014376A1

Description

Technical Field

The present invention relates to methods and assays to quantitate the amount of the neuropeptide secretoneurin (SN), the formation of which is associated with heart failure (HF). The present invention relates to an immunoassay for determining the concentration of SN in a fluid sample. The invention presents an accurate immunoassay for improved SN detection, thus facilitating the use of SN as a biomarker for HF detection and monitoring. Further, the invention comprises a method for determining SN peptide concentration in a fluid sample comprising unknown amounts of SN and the precursor protein of SN, Secretogranin II (SGII).

Background Art

[0001] Heart failure (HF) is a syndrome caused by the heart being unable to pump sufficiently to maintain blood flow to meet the body’s needs. HF is the leading cause of hospitalization of people aged 65 years and older and is typically associated with long and frequent readmissions. Ventricular arrhythmias account for a large proportion of HF deaths. To date, a major challenge relates to the accurate assessment and monitoring of the risk in acute and chronic HF stages.

[0002] The chromogranin-secretogranin (granin) protein family plays an important role in cardiac pathophysiology and has potential to serve as cardiovascular biomarkers. Of particular interest is secretoneurin (SN), a neuropeptide consisting of 33 amino acids (SGII 182-214) generated from the endoproteolytic cleavage of its precursor secretogranin II (SGII, Sg2 or chromogranin C). SN is internalized into cardiomyocytes by endocytosis and directly interacts with calmodulin (CaM) and CaM-dependent protein kinase II δ (CaMKIIδ), thus inhibiting CaMKIIδ activity. SN-mediated CaMKIIδ inhibition reduces localized release of Ca<2+ >from the sarcoplasmic reticulum, also called Ca<2+ >sparks. Thus, SN directly influences cardiomyocyte Ca<2+ >handling (Ottesen A.H (2015)). SN levels are elevated in patients with HF compared to SN levels in healthy subjects. Release of SN into the circulation seems to be mediated by the neuroendocrine system and from damaged cardiomyocytes and myocardium. Circulating SN levels provide strong and complementary information to established biomarkers in patients with acute HF and in patients with ventricular arrythmia-induced cardiac arrest. Thus, SN represents a promising cardiovascular biomarker, and measuring circulating SN concentrations can be used as a tool for improved risk stratification of patients with HF. However, to have clinical potential as a biomarker one of the requirements is that there should be a robust, efficient, reliable, and easily available method for biomarker measurement. This is currently lacking and presents a challenge for fully exploring the clinical potential of SN as a cardiovascular biomarker. To date, there are several suggested procedures for measuring SN, including radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and biosensors. Noteworthy, RIAs and biosensors are not readily available thus only the ELISA-based procedure has found some level of commercial use. Common for all these methods is the employment of antibodies to bind the analyte, SN, in the sample being analyzed. However, the methods differ widely in terms of the solid phase used, the antibodies against SN, and the method of obtaining an output measure on SN levels. For example, non-commercial in-housedeveloped RIAs such as disclosed by others (Kirchmair R (1993), Stridsberg M (2008)) employ the principle of competitive binding between the SN analyte and radiolabeled SN or SN-analogues to an SN binding antibody. However, a key drawback with RIAs is the use of hazardous materials requiring special laboratory facilities due to radiation. Moreover, it is a time-consuming procedure and therefore impractical for daily routine use in clinical laboratories. As the methods utilize inhouse antibodies rather than commercially produced antibodies, these are not readily available for use in clinical applications. Most of the antibodies applied are polyclonal antibodies and are not sufficiently characterized. Thus, non-specific antibodies are used in procedures for measuring SN, such as the RIA applying an antibody reacting with free SN, precursor SGII and intermediate breakdown products (Kirchmair R (1993), McQueen D.S (1996)).

[0003] Another strategy used for determining the SN concentration in human samples makes use of the microtiter plate-based enzyme-linked immunosorbent assay (ELISAs) strategy, which is a basic application used to analyze soluble antigens (Myhre P. L., et al. (2021)). The method uses two SN-binding antibodies. One SN-binding antibody is coated onto a solid surface and binds and thus captures SN from the sample solution. The second SN-binding antibody binds to another region of SN. The second SN-binding antibody is conjugated to an enzyme label, which produces a measurable signal upon activation and hence allows detection of the bound SN analyte. The ELISA is a strategy omitting the challenges related to use of hazardous materials associated with RIA. However, the ELISA workflow consists of multiple steps including different incubations and several washing steps to remove excess reagents with different buffers. Moreover, the solid phase typically comprises microtiter plates made of polystyrene with low binding capacity and limited dynamic range. Other common drawbacks include limited sample throughput, manual handling steps, and cross-reactivity and specificity issues. ELISA is thus a laborious, lengthy, and error-prone method, working well for research, but not so well for more extensive studies and high throughput settings. Yet an alternative approach for determining the SN concentration in human samples involves magnetic particlebased assays employing a sandwich configuration, wherein one example of this technique used goat polyclonal antibodies to analyze SN (CN115109152A).

[0004] Because SN is produced by endoproteolytic cleavage of SGII, all structural elements of SN are present in SGII. Thus, potential cross-reactivity of antibodies directed towards SN (anti-SN antibodies) is likely. If used for analyzing SN concentration in a complex sample containing SN and SGII, anti-SN antibody cross reactivity will hamper the analytical output. If the sample contains low concentration of SN and high concentration of SGII, low SN antibody specificity presents a challenge due to competitive equilibrium reactions. In such scenario, if a sample is analyzed with i.e., ELISA using non-specific SN antibodies having cross-reactivity towards SGII, both SN and SGII in the sample will be bound by the SN antibodies. In such instances, low antibody specificity increases the risk of determining incorrect SN concentration in the sample and produces poor confidence test results.

[0005] Based on the above, to facilitate the use of SN as a biomarker, there is a need for new and improved analytical methods to detect SN concentrations robustly and easily in complex samples containing both SN and SGII. The improved analytical methods should mitigate the major challenges related to accurate assessment and monitoring of the risk in acute and chronic HF stages.

Brief description of the drawings

[0006] Figure 1 shows the 33-amino acid neuropeptide secretoneurin (SN), generated as a cleavage product of human secretoneurin II.

[0007] Figure 2 presents the proposed structure of SGII based on extracted structural simulation data from a publicly available repository (Uniprot/Alphafold) and SN from bioinformatic structural simulations conducted by the applicant.

[0008] Figure 3 provides a representation of the epitopes identified for a range of sheep a-SN monoclonal antibodies tested in a binding assay against 43 recombinant secretoneurin peptides. SNNT: N-terminal binding a-SN monoclonal antibodies, SNCT: C-terminal binding a-SN monoclonal antibodies.

[0009] Figure 4 provides a line graph of the signal intensity at 405nm absorbance for different concentrations [ng/mL] of sheep a-SN monoclonal antibodies reflecting the binding of these antibodies towards SGII coated on microplates.

[0010] Figure 5 provides a curve illustrating the binding of various sheep a-SN monoclonal antibodies against non-coated/free SGII in solution.

[0011] Figure 6 provides the standard curve obtained from a magnetic bead-based sandwich immunoassay representative for a defined set of parameters for measuring secretoneurin in unknown samples. The x-axis shows the concentration of the SN calibrators, the y-axis the luminescence signal.

[0012] Figure 7 provides the calibration curve obtained from a magnetic bead-based competitive immunoassay representative for a defined set of parameters for measuring secretoneurin in unknown samples. The x-axis shows the final concentration of SN after 1:1 dilution with acridine labelled MAB, which had a final concentration of 0.1 µg/mL in the preincubation mix.

[0013] Figure 8 provides the calibration curve obtained from a non-magnetic beadbased sandwich immunoassay representative for a defined set of parameters for measuring secretoneurin in unknown samples.

[0014] Figure 9a highlights the analytical signal of a magnetic bead-based sandwich immunoassay for determining secretogranin as a function of particle amount used.

[0015] Figure 9b shows the relationship between delta RLU (signal difference between high SN-calibrator and zero calibrator) and the amount of particles added.

[0016] Figure 10a demonstrates the assay performance of a particle-based sandwich immunoassay for analyzing secretoneurin in the preferred embodiment of the present invention.

[0017] Figure 10b provides the correlation with the assay of 10a with SN-ELISA.

Summary of invention

[0018] It is therefore an objective of the present invention to provide strategies in the form of an immunoassay and analytical methods which are useful for improving the specific detection of SN in complex fluid samples containing a mixture of unknown amounts of SN and SGII, to facilitate the use of SN as a biomarker. The present disclosure provides an immunoassay for determining SN peptide concentration in a fluid sample.

[0019] In a first aspect, the invention provides an immunoassay for determining secretoneurin peptide concentration in a fluid sample, the immunoassay comprising a mixture of

at least one monoclonal antibody with binding specificity exclusively towards secretoneurin and without cross-reactivity towards the precursor protein secretogranin II (SG II, chromogranin C) (a-SN MAB I);

a catching component, wherein the catching component is immobilized onto a solid phase;

a signaling component, wherein the signaling component comprises a label;

at least one assay buffer;

wherein the a-SN MAB I comprises CDRs of any of reference id: g-m, being amino acid sequences GLSLTS, AISRSGRTY, GYSGAEAINV, SGSSSNIGRGWGS, DATTRAS, YAWDSSSSDGL.

In one embodiment, the at least one monoclonal antibody with binding specificity exclusively towards SN and without cross-reactivity towards the precursor protein secretogranin II (a-SN MAB) comprises complementarity-determining regions (CDRs) selected from the group of reference id: g-m, as provided herein.

[0020] In an equal aspect, the invention provides a method for determining SN peptide concentration in a fluid sample from a subject. In one embodiment, the method comprises use of the immunoassay of the first aspect.

[0021] In one embodiment, the method for determining SN peptide concentration in a fluid sample, is a sandwich immunoassay method comprising a sequence of steps including to:

(1) Mix a fluid sample from a subject with sandwich immunoassay components, the immunoassay components comprising a mixture of:

a catching component comprising a monoclonal antibody (MAB I) with binding specificity exclusively towards SN and without cross-reactivity towards the precursor protein secretogranin II (SGII, chromogranin C),

a solid phase, wherein the MAB I is immobilized onto;

a signaling component, wherein the signaling component comprises a label linked to a monoclonal SN antibody (MAB II);

at least one assay buffer,

(2) Incubate the mix from step 1 for a short time period forming a MAB I-SN-MAB II sandwich,

(3) Separate and retain the MAB I-SN-MAB II sandwich from unbound components using a separator appropriate for the solid phase,

(4) Wash the retained MAB I-SN-MAB II sandwich with wash buffer,

(5) Optionally, activate the label on the MAB II.

In another embodiment, the immunoassay method is a competitive immunoassay method comprising a sequence of steps including to:

(1) Mix a fluid sample from a subject with immunoassay components comprising a mixture of:

a catching component comprising a monoclonal antibody with binding specificity exclusively towards SN and without cross-reactivity towards the precursor protein secretogranin II (SGII, chromogranin C) (MAB I) or an SN analog, wherein the catching component is immobilized onto a solid phase; a signaling component, wherein the signaling component comprises a label attached to an SN analog or a label attached to a MAB I;

at least one assay buffer,

(2) Incubate the mix from step 1 for a short time period forming a catching component-signaling component complex,

(3) Separate and retain the catching component-signaling component complex from unbound components using a separator appropriate for the solid phase,

(4) Wash the retained catching component-signaling component complex with a wash buffer,

(5) Optionally, activate the label on the signaling component.

Detailed description of the invention

[0022] SGII is a member of the granin protein family and in humans SGII is a protein consisting of 617 amino acids. Endoproteolytic cleavage of SGII gives rise to SN (SGII 182-214) which is a 33 amino acid long neuropeptide, please see Figure 1 herein. Human SN has an amino acid sequence of Formula I:

TNEIVEEQYTPQSLATLESVFQELGKLTGPNNQ (Formula I).

[0023] Formation of SN, or SN processing, is species- and tissue-specific. In humans, SN processing occurs in cardiomyocytes and neuroendocrine tissues. In the failing myocardium, the protease mediated SGII cleavage activity is increased, thus leading to increased levels of SN e.g., in the blood. SN is internalized into cardiomyocytes by endocytosis and directly interacts with calmodulin (CaM) and CaM-dependent protein kinase II δ (CaMKIIδ), thus inhibiting CaMKIIδ activity. SN-mediated CaMKIIδ inhibition reduces localized release of Ca<2+ >from the sarcoplasmic reticulum, also called Ca<2+ >sparks. Thus, SN directly influences cardiomyocyte Ca<2+ >handling. SN levels are elevated in patients with HF compared to healthy subjects and is an established biomarker for monitoring HF patients or for improved risk stratification. The invention provides a new immunoassay and an improved method for detecting SN and determining SN concentrations in complex samples containing unknown amounts of SN and the precursor protein of SN, SGII. The immunoassay can have a sandwich-based design format or a competitive design format. A sandwich assay using two antibodies generally provides higher specificity and sensitivity than a competitive immunoassay using one antibody. In a preferred embodiment, the immunoassay has a sandwich-based design format. The choice of immunoassay format depends on several factors, i.e., the availability of reagents needed and the required dynamic range for the particular immunoassay. A common feature for both immunoassay design formats is that they comprise at least one monoclonal antibody with binding specificity exclusively towards SN and without cross-reactivity towards the precursor protein SGII, herein called MAB I, or more specifically a-SN MAB I. Moreover, both immunoassay design formats rely on a catching component which is immobilized onto a solid phase, and a signaling component comprising a label. Together, these components provide a way of determining the SN concentration in a sample.

[0024] In one embodiment, the immunoassay has a sandwich-based design which provides a direct approach for detecting SN. The sandwich-based immunoassay uses two monoclonal antibodies with binding specificity towards different regions of the SN neuropeptide. One monoclonal antibody is highly specific towards SN and binds exclusively to SN without cross-reactivity towards the precursor protein SGII (a-SN MAB I). The other monoclonal antibody has binding affinity towards other regions of SN than MAB I, wherein the region can be the N-terminal region of SN or a central region of SN. However, this other monoclonal antibody may also bind to SGII (MAB II). When referring to the C- or N-terminal region of SN this equates to the terminal or end regions of the SN peptide molecule. The C- and N-terminal regions are separated by a central region of SN, wherein the central region of SN has an amino acid sequence of Formula II:

[0025] ATLESVF (Formula II).

[0026] As such, the C-terminal region of SN should be interpreted as the C-terminal region spanning to the central region of SN, and the N-terminal region of SN should be interpreted as the N-terminal region spanning to the central region of SN.

[0027] In one configuration of the sandwich-based immunoassay, the MAB I is immobilized onto a solid phase and serves as the catching component, and the MAB II is linked to a label and serves as the signaling component. In a second configuration of the sandwich-based immunoassay, the MAB II serves as the catching component and the MAB I serves as the signaling component. Because MAB I and MAB II bind to different regions of SN, the antibodies can form a binding sandwich comprising the MAB I, SN and MAB II. Since SN is produced by endoproteolytic cleavage of SGII all structural elements of SN are present in SGII and presents a challenge with potential cross-reactivity of anti-SN antibodies resulting in incorrect or inaccurate test results. However, by employing MAB I and MAB II, as a catching and signaling component in an appropriate manner, a quantitative amount of SN in the sample can be bound and labelled and later detected after washing the particles in washing buffer and reading the analytical signal.

[0028] In another embodiment, the immunoassay has a competitive assay design which provides an indirect approach of detecting SN. In contrast to the sandwichbased design a competitive immunoassay design relies on only one highly SN-specific antibody. Hence, the competitive immunoassay design uses a monoclonal antibody which is highly specific towards SN without cross-reacting towards the precursor protein SGII (a-SN MAB I). As further detailed below, preferably the MAB I binds exclusively to the C-terminal region of SN. Further, the competitive immunoassay design includes an SN analog. The SN analog is either the natural SN neuropeptide or a synthetic peptide which is structurally similar to the SN neuropeptide or fragments thereof. In one configuration of the competitive immunoassay, the catching component comprises the a-SN MAB I immobilized onto a solid phase, and the signaling component comprises the SN analog and a label. A fixed amount of the labeled SN analog and a variable, unknown amount of the unlabeled SN present in the sample (SN analyte) thus compete to bind to the a-SN MAB I.

[0029] In the second configuration, the catching component comprises the SN analog immobilized onto a solid phase, the SN analog is unlabeled. The signaling component comprises the a-SN MAB I and a label. The SN analog of the catching component and the unlabeled SN analyte present in the sample serve as competitive binding sites for the labeled SN MAB I. Hence, for both competitive immunoassay design configurations there is an inverse relationship between the amount of unlabeled SN analyte present in the sample and the signal intensity produced.

[0030] Hence, in a first aspect, the invention provides an immunoassay for determining SN peptide concentration in a fluid sample, the immunoassay comprising a mixture of:

at least one monoclonal antibody with binding specificity exclusively towards SN and without cross-reactivity towards the precursor protein secretogranin II (SG II, chromogranin C) (a-SN MAB I);

a signaling component, wherein the signaling component comprises a label; at least one assay buffer.

In one embodiment, the a-SN MAB I comprises CDRs selected from reference id: gm, as listed in Table 3 herein.

[0031] The immunoassay is based on monoclonal antibodies. Antibodies are glycoproteins produced and secreted by specialized B lymphocytes known as plasma cells. The general Y-shaped structure of antibodies comprises three regions, two antibody-binding domain fragments (Fab) and one Fc fragment. Antibodies are host proteins produced in response to molecules and organisms, which they ultimately neutralize and/or eliminate. The molecules stimulating antibody production are referred to as antigens. Antibodies are found in plasma and extracellular fluids and serve as the first line of response and comprise one of the principal effectors of the adaptive immune system. Normally, antibodies carry out two main roles. Firstly, antibodies bind to distinct regions of the antigen, referred to as the epitope. Each antibody arm has an epitope binding site, making each antibody molecule at least bivalent. Secondly, upon epitope binding, another part of the antibody (the constant region) mediates the effector functions which includes activation of immune cells or the complement system or induction of phagocytosis to neutralize or eliminate the antigen. Most antigens are highly complex, causing them to present numerous epitopes that are recognized by a large number of lymphocytes. Upon antigen recognition, each lymphocyte is activated to proliferate and differentiate into plasma cells, resulting in formation of polyclonal antibodies (PABs) which can bind to different antigen epitopes depending on the epitope which caused lymphocyte activation. In contrast, monoclonal antibodies (MABs) are antibodies produced by a single B lymphocyte clone. As a result, MABs are specifically recognizing only one epitope structure of the antigen. In other words, MABs are monospecific. In one embodiment, either of the MAB I and MAB II comprise the full MAB molecule comprising two Fabs and one Fc fragment. In another embodiment, either of the MAB I and MAB II comprise a fragment of the full MAB molecule wherein this fragment is one Fab fragment, alternatively two Fab fragments held together by disulfide bonds (Fab2).

[0032] The specificity of an antibody refers to its ability to recognize a specific epitope in the presence of other epitopes. An antibody with high specificity binds to a lower number of different epitopes than an antibody with low specificity, thus has a lower degree of cross-reactivity. The highest specificity is reached when an antibody binds only to one defined epitope. MABs, which specifically recognize only one epitope structure of the antigen to which they were raised, are inherently specific. The affinity of an antibody refers to a measure of the binding strength for a monovalent antibody. The interaction adheres to thermodynamic principles and is described by the affinity constant Ka, which describes the amount of antigen-antibody complex forming at equilibrium. Antibodies with high affinity bind larger amounts of antigen with a greater stability in a shorter time than those with low affinity and are preferrable for use in immunochemical techniques. Due to the heterogeneous nature of PABs comprising varying affinities, affinity can only be estimated for PABs.

However, it is possible to determine precise affinities for MABs due to their homogenous nature. In the present invention, the applicant has conducted experiments to determine the Ka of the SN MABs by surface plasmon resonance technology, please see Example 1 herein. The Ka of the SN MABs has been determined to be in the range of 1x10<7 >to 1x10<10 >as presented in Table 1. In one embodiment, suitable a-SN MABs have an affinity constant, Ka, in the range 1x10<7 >to 1x10<9>, more preferably the Ka is 1x10<9 >or above.

[0033] With respect to native protein antigens, the binding affinity of antibodies is generally influenced by conformational determinants. Conformational structure of a protein or peptide can be influenced by numerous factors, including association with other proteins, post-translational modification, and pH. Because MABs target a single epitope, this is of high importance. In contrast, PABs recognize a broader range of epitopes, some of which are likely to have a linear structure. As a results, PABs are not impacted to the same extent as MABs by conformational changes. The principal advantage of MABs over PABs is their homogeneity, monospecificity and consistency. The monospecificity offered by MABs provides useful for identifying single members of protein families. Because MABs are produced by fusing the B lymphocyte to a cancerous cell (myeloma cell), this allows for creating a hybridoma which subsequently enables production of the MAB as a constant and renewable resource. This offers great advantage over PABs which are generated using multiple animals introducing several factors of variation. Moreover, the quantity of PABs obtained is limited to the size of the animal and its lifespan. This can therefore offer challenges with reproducibility, which is of vast importance for diagnostic testing.

[0034] The process of MAB development includes successive working phases comprising; the generation of antigen-specific B cells, the fusion of these B cells with myeloma cells to form hybridomas, the cloning and selection of the specific hybridoma clone and the up-scaling of MAB production. Traditionally, the former and latter of the working phases listed involves the use of laboratory animals. Sheep demonstrate a higher immune sensitivity compared to many other commonly used animals for antibody production, such as rabbits, goats, mice, or chickens. This feature enables a wider range of epitope recognition than other host species. MABs produced from immunized sheep generally possess high antigen specificity and affinity compared to MABs obtained from other species. This makes sheep MABs particularly valuable for use for biomedical and diagnostic purposes. In one embodiment, the MABs of the immunoassay are selected from the group of mammalian species, for example from the group of rabbits, goats, mice, chickens, and sheep. In a preferred embodiment, the MABs of the immunoassay are from sheep. Hence, both the MAB I and the MAB II are sheep monoclonal antibodies.

[0035] In one embodiment, the immunoassay has a sandwich-based configuration. The sandwich immunoassay is based on a sandwich principle which has been an established molecular strategy for years. The ability of antibodies to selectively bind a specific epitope on a molecule such as a protein or peptide has been thoroughly exploited through the years and is being used for several research and clinical applications. The sandwich-based methodology uses one antibody for capturing the antigen of interest and another antibody for detection, both antibodies having binding specificity for the same antigen. Importantly, the antibodies recognize and bind different epitopes of the same antigen, meaning they bind to different regions of the same antigen. Thus, the antigen is “sandwiched” between the two antibodies. In the present invention, the at least one monoclonal antibody with binding specificity exclusively towards SN and without cross-reactivity towards the precursor protein secretogranin II (SGII, chromogranin C) is defined as MAB I, or more specifically as a-SN MAB I. The MAB I can be utilized in both the direct sandwich-based configuration and in the indirect, competitive configuration of the immunoassay. In the sandwich-based configuration of the immunoassay, the signaling component comprises a monoclonal a-SN antibody with binding affinity towards other regions of SN than SN MAB II and is defined as SN MAB I. The sandwich strategy offers advantages in terms of high specificity which makes it a suitable approach to analyze complex samples. Meanwhile, due to the need for two antibodies against the same antigen which recognize different epitopes and simultaneously work well together, this is a challenging assay design requiring many considerations for optimal performance.

[0036] Although most SN N- and C-terminal reacting MABs can pair and form a MAB I-SN-MAB II sandwich with each other, not all of them can. This is demonstrated in experiments conducted by the applicant wherein combinations of MAB I and MAB II pairs were tested for their compatibilities to form a MAB I-SN-MAB II sandwich complex, and to subsequently produce an analytical signal for quantification of SN concentrations in a sample, please see Example 2 herein. As shown in Table 2, most of the sheep a-SN N- and C-terminal reacting MABs tested can pair and form a sandwich (MAB I-SN-MAB II) with each other interchangeably when being used as catching and detection component. However, not all of them can. For example, the clone SNCT.3A7 was unable to form sandwich pairs with any of the other a-SN MABs tested. In one embodiment of the invention, the immunoassay enables determination of SN peptide concentration by forming a MAB I-SN-MAB II sandwich complex and producing an analytical signal for quantification of the SN concentrations.

[0037] In the sandwich-based configuration of the immunoassay, the catching component is immobilized on a solid phase with the function of binding or catching the free SN from the surroundings. Herein, the catching component can be MAB I or MAB II, most preferably MAB I. The signaling component which comprises a label further binds to SN at a region different to the region bound by the catching component. The signaling component can be MAB I or MAB II, most preferably MAB II. Thus, a sandwich is formed comprising the MAB I, SN and MAB II, wherein MAB I and MAB II are bound to different regions of SN. In one embodiment, the antibody configuration of the sandwich immunoassay is such that MAB I which has binding specificity exclusively towards SN and can specifically bind to SN without crossreacting with SGII is the catching component. In this configuration, the MAB II which has binding affinity to other regions of SN, such as the N-terminal region of SN is the signaling component. MAB II thus binds to a region of SN different than that of the MAB I.

[0038] Since SN is produced by endoproteolytic cleavage of SGII all structural elements of SN are present in SGII. Thus, potential cross-reactivity of anti-SN antibodies is likely. If used for analyzing SN concentration in a complex sample containing SN and SGII, anti-SN antibody cross reactivity will hamper the analytical output and produce a test result of poor confidence. This is particularly an issue in the event the sample contains low concentration of SN and high concentration of SGII due to competitive equilibrium reactions. In such scenario, if a sample is analyzed with a sandwich ELISA using non-specific SN antibodies having crossreactivity towards SGII, both SN and SGII in the sample will be bound by the SN antibodies thus increasing the risk of determining falsely high SN concentration in the sample. However, the risk of such erroneous test analysis is mitigated by utilizing an immunoassay of the invention wherein at least one MAB has binding specificity exclusively towards SN and without cross-reactivity towards SGII (MAB I).

[0039] Moreover, the applicant conducted bioinformatic structural simulations of SN and extracted structural simulation data of SGII from a publicly available repository (Uniprot/Alphafold) please see Figure 2 herein. The bioinformatic structural simulations of SN and SGII support that the amino acid sequence corresponding to the SN peptide sequence adapts a non-linear, bent, discontinuous conformation. The amino acid sequence corresponding to the SN N-terminal is in a more open loop structure, whereas the amino acid sequence corresponding to the SN C-terminal region is in a helical structure buried deeper into the SGII protein structure. Although the model confidence for some regions of SGII is relatively low, the structural simulations support the view that a difference in conformation of the C-terminal part of SN in SG II compared to the free C-terminal part in the SN peptide in solution can explain the selectivity of the a-SN C-terminal binding MABs. Accordingly, in one embodiment the antigen-binding domain (Fab) regions of the SN MABs have a threedimensional conformation matching the structural configuration of the SN antigen so that it facilitates binding between the SN antigen and the Fab regions of the SN MABs.

[0040] The immunoassay is for determining SN peptide concentration in a fluid sample. Suitable fluids are fluids from a subject, particularly human subjects, likely to comprise SN. Suitable fluids are blood, in the form of serum or plasma, or other body fluids such as cerebrospinal fluid. Common for all samples is that they are complex and contain unknown concentrations of SN and SGII. The required sample volume is dependent on factors such as the concentration of the MAB I-coated bead particles, the MAB II containing a label and the instrument used to run the assay. The required sample volume is in the range of 2-100 µL, such as typically 2-10 µL, 10-50 µL or 50-100 µL, alternatively 5-15 µL or 20-60 µL, alternatively 10-30 µL, more preferably about 10 µL.

[0041] The catching component of the immunoassay is immobilized onto a solid phase. Such solid phase is provided in the form of superparamagnetic bead particles (magnetic beads) or in the form of non-magnetic bead particles. In one embodiment, the solid phase of the immunoassay is comprised of magnetic beads. Magnetic beads (or superparamagnetic bead particles) are one of the most versatile tools in molecular biology for easy and effective isolation of biomolecules. Magnetic beads are typically made up of tiny (20 to 30 nm) particles of iron oxides, such as magnetite (Fe3O4) and maghemite (gamma-Fe2O3), which give them superparamagnetic properties. Unlike more common ferromagnets, superparamagnetic beads exhibit magnetic behavior only in the presence of an external magnetic field. The magnetic beads may be made of a magnetic particulate material and a polystyrene shell which encases the magnetic particulate material. The magnetic material may comprise magnetite (Fe3O4), maghemite (gamma-Fe2O3) or a mixture of these which affects the iron (Fe) content of the particles. The polystyrene shell which encases the magnetic material may be functionalized with functional groups which provide a defined surface chemistry of the particle. The magnetic bead particle surface chemistry depends on the particle surface properties and functional groups incorporated onto the bead particle surface. The magnetic beads typically comprise a hydrophobic surface, alternatively a hydrophilic surface.

[0042] The magnetic bead particles are preferably monosized, with a magnetic bead particle size in the range of 0.5-5 µm, such as typically 0.5-3 µm or 3-5 µm, alternatively 2-4 µm, more preferably about 3 µm. The bead particle size is an important feature for the performance of the sandwich immunoassay. A smaller bead particle diameter secures higher binding amounts of the MAB I per particle mass, due to the increased surface area of a bead particle of smaller diameter compared to a larger diameter bead particle. However, the bead particle diameter affects the separation time, i.e., the time it takes to separate the magnetic beads containing the MAB I-SN-MAB II sandwich from the reaction mixture. This separation is necessary to allow excess reactants to be removed before washing the particles containing the MAB I-SN-MAB II sandwich. A larger bead particle diameter gives a faster separation time, thus contributes to reduce the total assay time. Accordingly, the bead particle diameter of the immunoassay is optimized with regards to both binding properties and separation time.

[0043] In another embodiment, the immunoassay comprises a solid phase comprised of non-magnetic bead particles (non-magnetic beads). The non-magnetic beads typically have a bead particle size in the range of 100-5000 nm, such as typically 100-200 nm, 200-500 nm, 500-1000 nm, alternatively 1000-5000 nm. The preferred bead particle size is dependent on factors such as the immunoassay design and the mode of separation. If the immunoassay is a flow through immunoassay using filtration as mode of separation, the preferred bead particle size is > 250 nm. If the immunoassay comprises lateral flow systems with stop zones to immobilize or capture bead particles, the preferred bead particle size is > 400 nm. If the immunoassay uses centrifugation as mode of separation, the preferred bead particle size is >500 nm.

[0044] The type of solid phase used in the immunoassay, i.e., magnetic or nonmagnetic bead particle governs the method of separation of the bead particles containing the catching component with SN bound thereto from the surrounding reaction mixture. In the case the solid phase used in the immunoassay is magnetic bead particles, a magnetic separator is used for separation. In the case the solid phase used in the immunoassay is non-magnetic bead particles, a membrane or a filter is used for separation by an external force. The membrane or filter may comprise nitrocellulose, paper, glass, nylon or polyethersulfone. The external force may comprise capillary forces, vacuum or centrifugation.

[0045] The bead particle surface of the solid phase has a defined surface chemistry comprising functional groups. The functional groups enable immobilization of the catching component, MAB I or the MAB II to the solid phase, i.e., by linking functional groups of the bead particle surface to functional groups of the MAB I or the MAB II. The defined surface chemistry of the bead particle surface must therefore be adapted to be compatible with the functional groups available on the MAB I or the MAB II. Thus, in addition to the importance of the bead particle spherical shape and size, the defined surface chemistry of the bead particle surface is important for minimizing chemical agglutination and non-specific binding onto the bead particle surface.

Streptavidin-biotin chemistry is a commonly used method to immobilize the catching component onto the solid phase surface in a variety of immunoassays. However, since biotin supplementation has expanded significantly over the years as prescribed therapies, and high dose biotin “health supplements” promoted for their supposed benefits are freely available without prescription, very high biotin concentrations can be found in patient samples. Thus, biotin is posing a significant challenge to many streptavidin-based immunoassays leading to incorrect test results and thereby to inappropriate patient management and misdiagnosis. Hence, the surface chemistry used to immobilize the catching phase to the solid phase is critical for the performance of the assay. In one embodiment, the functional groups of the bead particle surface are selected from hydrophobic groups, such as toluene sulfonyl groups (tosyl groups), tresyl, chloromethyl, aldehyde, or alternatively from hydrophilic groups such as epoxide groups, carboxylic acid groups, and surface amino groups, or alternatively antibody binders including Protein A, Protein G or a-sheep IgG antibodies. In some embodiments the functional groups of the bead particle surface are either tosyl groups or epoxide groups.

[0046] The functional groups of the bead particle surface provide different degree of conjugation of the catching component onto the bead particle surface, wherein the catching component can be MAB I or MAB II, most preferably MAB I. The conjugation of MAB I or MAB II onto the bead particle surface via functional groups on the bead particle surface is referred to as loading. Importantly, loading is a function of particle surface area, again defined by particle size and particle and surface morphology, as well as the active chemical functionalities available for immobilizing proteins and ligands. Thus, the preferred loading capacity will vary depending upon the particle applied. Generally, the maximum loading a specific particle can provide should be utilized as the most preferred embodiment. Tosyl groups typically bind 10-15 µg IgG per mg beads (of 2.8 µm diameter) and epoxide groups typically bind 5-10 µg IgG per mg beads (of 2.8 µm diameter). A good loading onto the bead particle surface is crucial for the specificity and sensitivity of the sandwich immunoassay performance because a good surface coverage of the bead particle with the MAB reduces the degree on non-specific binding of non-SN components to the naked bead particle surface. Moreover, a good loading of MABs onto the bead particle surface ensures that a lower number of magnetic bead particles can be used per sample. A consequence of this is that the sandwich immunoassay run time is reduced and thus the determination of SN concentrations from a patient’s fluid sample can be achieved faster. In the present invention, for a direct sandwich-based immunoassay design, a good loading onto the bead particle surface equates to a loading of > 10 µg MAB per mg bead particle (of 2.8 µm diameter). Accordingly, in one embodiment, the solid phase comprises bead particles, e.g. of 2.8 µm diameter, with a surface adapted to enable a loading of > 10 µg MAB per mg bead particle, such as 1-10 µg MAB per mg bead particle, typically 1-5 µg, 5-10 µg, and 10-15 µg MAB per mg bead particle, most preferably 8-15 µg MAB per mg bead particle.

[0047] For a competitive immunoassay design the loading requirement depends on the immunoassay design and are a bit more intricate than for a sandwich format. Using particles coated with an a-SN MAB I, and labelled SN or a labelled derivative thereof in solution, the particle loading must be balanced between analytical signal strength, dynamic range of the assay, and amount of sample volume, factors to a large extent controlled by the affinity of the antibodies used. Yet, an appropriate loading of MAB I for particles of 2.8 µm diameter size in a competitive setup has been found to be > 0.5 µg MAB I per mg of particles. For a competitive assay design where SN or an analogue thereof is coated on the particle using labelled a-SN MAB I in solution, the molar amount of SN or SN analogue must be balanced against the sample volume and the molar range to be covered by the assay.

[0048] In either case, immobilized MAB I or labelled MAB I has to be balanced against its respective reactants to promote competition towards binding to the antibody.

[0049] The loading requirements when using non-magnetic beads corresponds to the loading of a magnetic particle of the same size. However, for the sandwich approach it is generally preferred to utilize the larger loading capacity of small diameter beads compared to larger particles. For competitive set-ups using submicron sized particles, the same general requirement of reactant concentration and ratio apply as with magnetic beads to secure and promote competitive binding towards the antibody.

[0050] Optionally, as an alternative to a high particle loading, the number of particles used per immunoassay may be increased. This is especially relevant in the case that particles have a low particle loading.

[0051] Tosyl groups typically bind the MAB via the more hydrophobic amino groups in the antigen-binding domain (Fab domain) region, which may provide a challenge with respect to keeping antigen binding properties of the MAB unchanged. With regards to this feature, epoxide groups are advantageous to use because they provide good orientation of the coupled MAB. Moreover, epoxide groups have extremely low non-specific binding of proteins and dyes, thus contributing to the specificity and sensitivity of the sandwich immunoassay.

[0052] The signaling component comprises a label. The label can comprise one or more of fluorophores selected from the group of fluorescein, lanthanide chelates, europium chelates, resorufin (oxazines), phthalocyanines, cyanine dyes, luminophores such as substituted isoluminol, acridinium esters, electrochemiluminophore such as ruthenium, enzymes, or particles such as dyed or fluorescent latex particles, colloidal gold, and europium chelate nanoparticles. In one embodiment, the label is selected from the group of acridinium esters, such as 3-[9-[4-(2,5-dioxopyrrolidin-1-yl)oxycarbonyl-2,6-dimethylphenoxy]carbonylacridin-10-ium-10-yl]propane-1-sulfonate (NSP-DMAE-NHS) and 3-(9-((4-((23-((2,5-dioxopyrrolidin-1-yl)oxy)-19,23-dioxo-3,6,9,12,15-pentaoxa-18-azatricosyl)carbamoyl)-2,6-dimethylphenoxy)carbonyl)acridin-10-ium-10-yl)propane-1-sulfonate (NSP-DMAE-HEG-Glu-NHS), enzymes such as horse radish peroxidase (HRP), alkaline phosphatase (ALP), and cyanine dyes such as Cy3/Cy5 or Cy7. In one embodiment the label is selected from the group comprising alkaline phosphatase, horseradish peroxidase, fluorescein, lanthanide chelates, acridine esters, luminol, luminol derivatives, ruthenium complexes, europium chelates, phtalocyanines, oxazines, latex particles, and colloidal gold.

[0053] Thus, the immunoassay offers a great flexibility with regards to the choice of label type to be part of the signaling component. The label type will depend on the immunoassay design applied and is to a large extent dependent on the immunoassay platform used for conducting the analysis. If the label comprises a luminophore, such as an acridine label, a chemiluminescent analytical signal is provided in the presence of H2O2 at alkaline pH that can be recorded typically at 420 nm. For other luminophores, e.g., luminol, luminescence is recorded typically at 425 nm, for europium chelates, typically at around 620 nm. The label can comprise a fluorophore such as europium chelates, phtalocyanines or oxazines, provided the immunoassay platform has the features needed to excite the fluorophores and correspondingly record the emitted fluorescent signal.

[0054] The signaling component comprises a label, wherein the signaling component comprises the MAB I or the MAB II, more preferably MAB II. As described in the section of immobilizing the catching component onto a solid phase, the same considerations must be taken regarding linking the MAB I or the MAB II to a label. In the case the signaling component comprises MAB II, the MAB II must be leniently labelled such that the antibody immunoreactivity, e.g., the ability of MAB II to bind to SN, is left unchanged. At the same time the MAB II must be labelled to a degree for it to produce an analytical signal sufficiently strong to secure low imprecision when the analytical signal is being read by the instrument. In one embodiment, the label is chemically bound to the MAB I or MAB II, referred to as conjugation. Conjugation of the label to the MAB II occurs by linking available functional groups of the MAB II to functional groups of the label. In one embodiment, the functional groups of the MAB II for conjugation to the label is selected from the group of free primary amine groups (-NH2), sulfhydryl groups (-SH), and carbohydrates (sugars) containing cis-diols which can be oxidized to create active aldehydes (-CHO). Moreover, functional groups suitable for subsequent conjugation may be introduced in the MAB II through derivatization of existing functional groups within the MAB II. Particularly, -SH groups may be introduced by modifying amine groups with N-succinimidyl S-acetylthioacetate (SATA). The choice of the functional group of the MAB II used for conjugating the label largely depends on which groups are available on the MAB II to achieve sufficient degree of labelling of the MAB II.

[0055] The immunoassay comprises at least one buffer. The at least one buffer has at least the function as an assay diluent to achieve a balanced ratio of MAB I conjugated to magnetic bead particles, labelled MAB II, and liquid sample and contains components with the function to prevent non-specific binding. In one embodiment, the at least one buffer may be used as a stand-alone reagent component in the immunoassay. In another embodiment, the at least one buffer can be combined with other components of the immunoassay such as the MAB I or the bead particles. In one embodiment, the at least one buffer is selected from the group of phosphate buffers, carbonate buffers, Tris-buffers, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), Bis-Tris, PIPES, MOPS, DIPSO, HEPPSO, HEPPS, tricine and bicine. Due to their volatility, carbonate and carbonate/bicarbonate buffers are less preferred options as the at least one buffer. The at least one buffer may also include detergents, such as e.g., Tween-20 and Pluronic F-127. The at least one buffer has a pH in the range of 4.0-9.0. In one specific embodiment the at least one buffer is comprised of e.g., phosphate buffer pH 7.4, NaCl, bovine serum albumin (BSA), non-specific IgG, detergent e.g., Tween-20. Specifically, the at least one buffer may comprise 10 mM phosphate buffer pH 7.4. Further the buffer may comprise 150 mM NaCl, 0.5 mg/mL BSA, 1 µg/mL non-specific sheep IgG, with detergent (e.g., Pluronic F-127) as used in studies by the applicant.

[0056] The standard curve of each immunoassay design format is demonstrated for a defined set of parameters considering bead particle type, bead particle size; bead particle surface chemistry; particle loading, and signal component labeling in Example 5. This includes the magnetic bead-based sandwich immunoassay (Example 5i, Figure 6), the magnetic bead-based competitive immunoassay (Example 5ii, Figure 7), and the non-magnetic bead-based sandwich immunoassay (Example 5iii, Figure 8). Moreover, the effect of increased amounts of bead particles in a bead-based sandwich immunoassay design is demonstrated (Example 6, Figure 9).

[0057] The immunoassay is based on monoclonal antibodies. Structurally, antibodies are composed of four polypeptides comprising two heavy chains and two light chains. The two identical copies of a heavy (~55 kD) and a light (~25 kD) chain are held together by disulfide and non-covalent bonds, together forming a molecule often represented by a schematic Y-shaped molecule of ~150 kD. The amino termini of the light and heavy chains associate to form an antigen-binding domain (Fab), which are located at the tip of each arm of the Y-shaped antibody structure. The carboxy terminal regions of the two heavy chains fold together to form the effector domain (Fc). Light chains comprise a variable amino terminal moiety of 110 amino acids and a constant region of similar length. Meanwhile, heavy chains comprise one variable region and at least three constant regions, each approximately 110 amino acids long. The variable regions of the light and heavy chain are responsible for forming the Fab domains, and the constant regions provide structural framework. Each variable region contains three hypervariable regions of 5-10 amino acids organized in loop structures, which make up the actual epitope binding sites or complementaritydetermining regions (CDRs) of the antibody. Each CDR is evenly distributed between four less variable framework (FR) regions. Thus, the CDRs provide a specific antigen recognition site at the amino termini of the antibody, and it is the hypervariability of these regions which enables antibodies to recognize an almost unlimited number of antigens.

[0058] In one embodiment, the MAB I of the immunoassay belongs to a MAB family of which the antibodies all have binding specificity towards the C-terminal part of SN, more specifically towards the amino acid sequence of Formula III FQELGKLTGPNNQ located at the C-terminal part of SN. One example of said antibody with binding specificity towards Formula III is the SN MAB clone SNCT.6B12, which comprises CDR regions of reference id g-m as shown in Table 3 below.

In a preferred embodiment, the at least one monoclonal antibody with binding specificity exclusively towards SN and without cross-reactivity towards the precursor protein secretogranin II (a-SN MAB) comprises CDRs selected from the group of reference id: g-m.

[0059] Table 3: CDR sequence information for the SN MABsSNNT.6D2 and SNCT.6B12 described herein.

[0060] The MAB family having specificity towards the C-terminal part of SN includes antibodies isolated from the clone SNCT.6B12. Existing assays based on monoclonal antibodies for specifically analyzing SN in human fluid samples including blood and plasma include the research-use-only SN ELISA assay (Myhre P. L., et al., 2021). This assay utilized a streptavidin-coated microplate to which biotinylated SN-binding primary MABs are conjugated and bind SN at the N-terminal region (SN MAB clone SNNT.2E8). Further in the ELISA assay, an HRP-conjugated SN MAB is added to achieve signal and thus quantitation of SN. The HRP-conjugated SN MAB binds the C-terminal region of SN (SN MAB clone SNCT.3B7). However, as part of characterizing potential cross-reactivity of their SN MABs towards the precursor protein SGII, the applicant conducted an ELISA-based binding assay using recombinant SGII as the ligand coated on microtiter plates (Example 4i). Surprisingly, they revealed that all SN MABs with binding specificity towards the N-terminal region also bind to SGII. In other words, the binding of the N-terminal binding SN MABs tested is not specific towards SN alone, but these SN MABs also bind to their corresponding epitopes present in SGII. On the contrary, none of the C-terminal binding SN MABs tested, including SNCT.6B12, showed cross-reactivity with SGII as seen in Figure 4. This is a surprising property of the tested SN C-terminal binding SN MABs, since SGII contains the complete SN structure as part of its amino acid sequence. Yet, the SNCT.6B12 can still differentiate between C-terminal SN and the same structural features in SGII by not binding the latter. One exception was the SN MAB clone SNCT.3A7, which showed cross-reactivity with SGII.

[0061] The cross-reactivity towards SGII was assessed for clone SNCT.3A7 using free SGII in solution (Example 4ii, Figure 5). This was to exclude the possibility that the cross-reactivity of clone SNCT.3A7 observed in Example 4i) was a consequence of conformational changes of SGII induced when coating SGII on microtiter plates. The results from Patent example 4 ii) using free, non-coated SGII shows that the sheep monoclonal a-SN antibody 3A7 as a representative for the SGII binding MABs tested in Example 4i) as well as the polyclonal a-SN antibody, also binds free, noncoated SGII. This shows that these a-SN antibodies also cross-react with free SGII, confirming that the results in Example 4i) are valid and not due to any conformational related changes induced by coating SGII to the microtiter plate, thereby exposing the binding epitopes in such a way that the antibodies can bind.

[0062] Bioinformatic simulations of SN/SGII conducted by the applicant supports that the amino acid sequence corresponding to the SN peptide is non-linear, and rather adapts a bent, discontinuous conformation (Figure 2). The structural properties of the SN epitope were investigated by the applicant through epitope mapping, please see Example 3 herein. A total of 43 peptides were used to map the binding epitope of different SN MABs. The members of each family, i.e., C- and N-terminal SN MABs bind to the same epitope, yet there seems to be no common characteristics with regards to amino acid sequences, as presented in Figure 3. The SN epitope mapping results support that SN in free state adapts a discontinuous conformation, as suggested by the bioinformatic structural simulations of SN/SGII. Moreover, the binding epitope for SNCT.3A7 which showed cross-reactivity with SGII is located in the central region of SN rather than in the SN C-terminal region (Example 3, Figure 3). Therefore, it seems more appropriate to consider clone SNCT.3A7 to be a SN central-region binding SN MAB rather than a C-terminal binding SN MAB.

[0063] Accordingly, in one embodiment the SN MABs with binding specificity towards the SN C-terminal region comprise one antibody family which can be used as a catching component or as a signaling component. In another embodiment, the SN MABs with binding specificity towards the SN N-terminal region comprises another antibody family which can be used as a catching component or a signaling component. Critical for the invention is that the antibodies used as catching component and signaling component cannot be members of the same antibody family.

[0064] In addition to properties of the SN epitope, there might be factors other than the structural availability of the N- and C-terminal domains of SN which influence the binding specificity of the SN MABs. For example, it could be common features in the CDRs of the SN MABs with binding affinity to the N-terminal and C-terminal (C-terminal binding SN MABs family), respectively, which account for the distinctive binding properties observed for these two SN MAB families.

[0065] Although the CDRs provide a specific antigen recognition site at the amino termini of the antibody, it may be that the FRs which are spaced between each CDR, offer a contribution to the antibody binding properties. FRs that affect antigen binding can be divided into two categories, depending on their contribution. The first category comprises FR residues that directly contact the antigen, thereby being direct part of the binding site. These FR residues can be located very close to the CDRs.

Alternatively, they can be located far from the CDRs in sequence yet be in close proximity to the CDRs in the three-dimensional structure. The second category comprises FR residues that are not directly in contact with the antigen but affect antigen binding indirectly. These FRs may also be either in close spatial proximity or distant to the CDRs. FRs in close proximity to the CDRs are assumed to provide a structural support to the CDRs by enabling the CDRs to adapt a correct orientation and conformation thus shaping the binding-site needed for antigen binding. FRs located spatially distant from the CDRs may affect the antigen binding site by directing the relative orientation of the variable regions of the light and heavy chain of the Fab domain. However, it is likely that the FRs offer a functional contribution to the observed specificity of C-terminal binding SN MABs. Accordingly, in one embodiment the CDRs and FRs of the SN MAB Fab regions, contribute to form a threedimensional conformation matching the structural configuration of the SN antigen so that it facilitates binding between the two. In another embodiment, the CDRs of the SN MAB contributes to the SN antigen binding. In another embodiment, the FRs of the SN MAB contributes to the SN antigen binding.

[0066] Some examples of CDR amino acid sequences of SN MABs with binding specificity to the SN C-terminal region or to the SN N-terminal region are listed in Table 3.

[0067] In another aspect, the invention provides a method for determining SN peptide concentration in a fluid sample from a subject. In one embodiment, the method comprising a sequence of steps including to:

mix the fluid sample with sandwich immunoassay components (step 1) to form a mixture,

incubate the mixture from step 1 for a short time period forming a MAB I-SN-MAB II sandwich (step 2),

separate and retain the MAB I-SN-MAB II sandwich from unbound components using a separator appropriate for the solid phase (step 3)

wash the retained MAB I-SN-MAB II sandwich from step 3 with wash buffer (step 4),

optionally, activate label (step 5).

The sandwich immunoassay components of step 1 comprise a mixture of;

a MAB I immobilized onto a solid phase wherein the MAB I has binding affinity to the C- or N-terminal region of SN (reagent a),

a MAB II linked to a label wherein the MAB II has binding affinity to the C- or N-terminal region of SN which is opposite to that of the MAB I (reagent b), and an assay buffer (reagent c).

The sandwich immunoassay component reagents a-c are defined as assay-specific reagents. In one embodiment the assay-specific reagents a-c and the fluid sample is mixed in step 1. This is the preferred embodiment of the method for determining SN peptide concentration in a fluid sample. In some embodiments step 1 is subdivided into two steps in which two of the assay-specific reagents a, b and c, and the fluid sample are mixed in step 1, followed by a step 2 wherein the third assay-specific reagent is mixed with the composition from step 1. Thus, step 1 can be mixing of reagents a, b and the liquid sample followed by step 2 wherein reagent c is added to the composition from step 1. Alternatively, step 1 can be mixing of reagents a, c, and the liquid sample, followed by step 2 wherein reagent b is added to the composition from step 1. The embodiments listed are examples of, but not limited to, alternative method steps for determining SN peptide concentration in a fluid sample. These are more laborious as compared to the most preferred embodiment, and as such are less preferred. However, in case of less optimal formulations of the assay-specific reagents a, b and/or c, there may be a need for optimizing the binding of SN in the liquid sample to MAB I (reagent a), alternatively to bind SN in the liquid sample to the MAB II (reagent b). In such events the listed, or other alternatives of conducting step 1 of the method may be necessary. The assay performance of a particle-based sandwich immunoassay for analyzing secretoneurin in the preferred embodiment of the present invention is presented in Example 7, Figure 10, Table 4, and Table 5.

[0068] The method for determining SN peptide concentration in a fluid sample comprises an incubation of the mixed assay-specific reagents and the liquid sample (step 2) for a short time period forming a MAB I-SN-MAB II sandwich. The short time period is in the range of 5-40 minutes, such as typically 5-10 minutes, 10-20 minutes or 20-40 minutes. In one embodiment, the short incubation period is typically about 10 minutes. The incubation time is determined by the specifications of the immunoassay platform used to conduct the immunoassay, as well as the affinity (Ka) of the MABs being used.

[0069] The assay time is typically around 10-15 minutes for a complete assay sequence and comprise; pipetting the reagents, mixing, incubation and reading the final analytical signal. However, the assay time depends on factors such as the incubation time of step 2 and can be up to 50 minutes. The analytical signal of a sample is subsequently compared against the corresponding analytical signal of a proper SN calibrator to calculate the final concentration of SN in the sample being analyzed. If automated the assay time can be significantly shorter than 10 minutes, around 5-8 minutes. This will to a large extent depend upon the features and abilities of the immunoassay platform used and how fast it can transfer the reagents, mix, and start the reaction as well as record the analytical signal. It will also depend on the affinity (Ka) of the MABs being used. Longer assay times can also be applied, e.g., 20-30 or 40-50 minutes. However, longer assay times will affect the throughput and can also negatively affect assay performance, e.g., assay imprecision, due to the increased background signal noise at prolonged incubation times.

[0070] In another aspect, the invention relates to the method for determining SN peptide concentration in a fluid sample from a subject, the method comprises using an immunoassay as disclosed in the first aspect.

[0071] In one aspect, the invention provides a method for determining SN peptide concentration in a fluid sample from a subject.

[0072] In one embodiment, the method comprises the use of a sandwich immunoassay comprising a mixture of:

a solid phase, wherein the MAB I is immobilized onto;

at least one assay buffer.

[0073] In another embodiment, the method comprises the use of a competitive immunoassay comprising a mixture of:

a catching component comprising a monoclonal antibody (MAB I) with binding specificity exclusively towards SN and without cross-reactivity towards the precursor protein secretogranin II (SGII, chromogranin C) or an SN analog, a solid phase wherein the catching component is immobilized onto;

a signaling component, wherein the signaling component comprises a label attached to an SN analog or a label attached to a MAB I;

at least one assay buffer.

[0074] The embodiments and features described in the context of one aspect, e.g., for the aspect directed to the immunoassay in any of the mentioned configurations, also apply to the other aspects of the invention, such as in the method for determining SN concentrations in a liquid sample containing unknown amounts of SN and SGII. The varieties of compounds and active substance options, and combinations, disclosed for one aspect hence also apply to the other aspects.

[0075] References:

Ottesen AH, Louch WE, Carlson CR, Landsverk OJB, Kurola J, Johansen RF, Moe MK, Aronsen JM, Høiseth AD, Jarstadmarken H, Nygård S, Bjørås M, Sjaastad I, Pettilä V, Stridsberg M, Omland T, Christensen G, Røsjø H. Secretoneurin is a novel prognostic cardiovascular biomarker associated with cardiomyocyte calcium handling. J Am Coll Cardiol.2015 Feb 3;65(4):339-351.

Kirchmair R, Hogue-Angeletti R, Gutierrez J, Fischer-Colbrie R, Winkler H (1993) Secretoneurin – a neuropeptide generated in brain, adrenal medulla and other endocrine tissues by proteolytic processing of secretogranin II (chromogranin C). Neuroscience 53:359–365.

Stridsberg M, Eriksson B, Janson ET (2008). Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours. Regulatory Peptides 5;148(1-3):95-8.

McQueen, D.S., Eder, U., Timm, M., Winkler, H., Dashwood, M.R., Bond, S.M.

(1996). Secretoneurin. In: Zapata, P., Eyzaguirre, C., Torrance, R.W. (eds) Frontiers in Arterial Chemoreception. Advances in Experimental Medicine and Biology, vol 410. Springer, Boston, MA.

Myhre PL, Ottesen AH, Faaren AL, Tveit SH, Springett J, Pyylampi J, Stridsberg M, et al., Performance of a Novel Research-Use-Only Secretoneurin ELISA in Patients with Suspected Acute Coronary Syndrome: Comparison with an Established Secretoneurin Radioimmunoassay. Cardiology, 2021.

Yuan G, Chen H, Xia C, Gao L, Yu C. Ultrasensitive electrochemical detection of secretoneurin based on Pb(2+)-decorated reduced graphene oxide-tetraethylene pentamine as a label. Biosens Bioelectron.2015 Jul 15;69:95-9.

Examples

[0076] Example 1: Determination of the Ka/Kd of the SN MABs by surface plasmon resonance technology.

[0077] The affinities and specificities of monoclonal antibodies were evaluated by surface plasmon resonance using a Biacore® instrument system (Cytiva, Sweden).

1) Immobilization of sheep monoclonal antibodies (MAB) and secretoneurin on dextran coated CM5 chip:

The carboxylated surface of the dextran chip was activated according to protocol supplied by the manufacturer. In short, 0.05M N-hydroxysuccinimimide/0.2M N-ethyl-N’-[3-(dimethylamino)propyl]carbodiimide hydrolchloride was injected over the carboxylated chip at 10 µL/min for 7 min, then 50 µg/mL MAB in 10 mM acetate, pH 4.8 was injected at 10µL/min for 7 min, and finally the chip was blocked by injection of 1 M ethanolamine at 10 µL/min for 7 min. The immobilization of secretoneurin on the dextran coated chip was performed in the same way.

2) Binding of MABs to secretoneurin coated dextran chip:

MABs were diluted in 0.01M hepes-buffer, pH 7.4, 0.15M, 0.003 M EDTA and 0.005% (v/v) Surfactant P20 (HBS-EP) to different concentrations and injected over the chip with immobilized secretoneurin at 5 µL/min for 3 min. The binding of monoclonal antibodies was followed in real time and the amount of mass bound is recorded in Refractive Units (RU) in the sensorgram. The on- and off-rates of binding were recorded for different concentrations of MAB and the affinity constants estimated (Table1)

3) Binding of MABs to secretoneurin prebound to non-overlapping MAB immobilized on dextran chip: the direct immobilization of the 33 amino acid peptide, secretoneurin on the chip may mask MAB specific epitopes. The binding of the different MABs to secretoneurin were therefore also determined with secretoneurin bound not directly to the chip but to a first MAB immobilized on the chip. Only MAB pairs that were non-overlapping were investigated (see Example 3).

Table 1. Affinity constants as measured by plasmon resonance

*27-fold greater deposition MAB1

The affinities were determined under conditions where the SN-peptide is under limiting conditions. So that no or very little rebinding of lost detection MAB will occur. The 100-fold greater apparent affinity observed when using excess capture MAB demonstrates the importance of the loading capacity of the substrate for the capture MAB.

[0078] Example 2: Ability of a-SN MABs to form MAB I-SN-MAB II sandwich pairs

1) A series of sheep a-SN monoclonal antibodies (a-SN MABs) were biotinylated at a biotin:antibody ratio of 2:1 by following standard conjugation conditions.

2) Nunc Maxisorp plates were coated for 90 minutes at 37°C by adding 100 µL per well of a series of a-SN MABs at 5 µg/mL diluted in 10 mM phosphate buffer, 0.150 M NaCl, pH 7.4 (PBS).

3) The plates were then blocked for 60 minutes at room temperature by adding 200 µL of 10 mg/mL BSA in PBS per well.

4) 100 µL per well of secretoneurin, 0.1 ng/mL in PBS was then added and the plates incubated on a shaker for 60 minutes at room temperature.

5) 100 µL of each of the biotinylated a-SN MABs prepared in step 1 (at 1.25 ng/mL in PBS) was then added to wells coated with different MAB pair candidates and the plates incubated on a shaker for 45 minutes at room temperature.

6) 100 µL per well was applied of streptavidin-HRP conjugate (Millipore) diluted 1:10000 in PBS and the plates incubated on a shaker for 45 minutes at room temperature.

7) 150 µL of TMB HRP -substrate (Kem-En-Tek) was then added per well, before incubating the plates for 10 minutes at room temperature.

8) 50 µL of stop solution, 0.5M H2SO4, was then added per well and the signal read at 450nm.

9) The absorption signals achieved from the various combinations of biotinylated a-SN MABs and a-SN MABs coated on the plates were used to identify the antibodies able to form a MAB I-SN-MAB II pair, and the strength of this formation.

In between each step 2-6 an automated plate washer was used to do 3 x 300µL/well washes using PBS 0.05% Tween 20. The same procedure was used in step 7, but 6 wash cycles was used instead of 3.

[0079] Table 2 presents the ability of different a-SN MABs to form sandwich pairs with each other.

[0080] Table 2: Table showing the ability of different a-SN sheep monoclonal antibodies to form MAB I-SN-MAB II sandwich pairs. A(450nm) = 0.02 represents the background signal.

[0081] <(*) >The N-terminal SN binding monoclonal antibody 4E2 was inactivated by biotinylation.

[0082] Example 3: Secretoneurin (SN) overlapping peptides epitope mapping of sheep a-secretoneurin monoclonal antibodies (sheep a-SN MABs)

[0083] 1) The following 43 peptides (all biotinylated at the N-terminal amino acid) were synthesized (representing fragments of the primary structure of secretoneurin (SN) in bold):

2) Nunc Maxisorp plates were coated overnight at 2-8°C adding 100 µL per well of streptavidin-HRP (Sigma) at 1µg/mL diluted in 10mM phosphate buffer, 0.150 M NaCl, 0.1% azide, pH 7.4 (PBS).

3) The plates were then blocked with 200 µL of milk powder 20 mg/mL in PBS per well for > 30 minutes at 37°C.

4) 100 µL of each of the 43 peptides from step 1 diluted to 1 µg/mL in 10 mg/mL BSA/PBS were then added per well and incubated for 1hour at 37°C.

5) 100 µL of each of the sheep a-SN monoclonal antibodies to be tested (at 1 µg/mL in PBS) were then added per well to each of the 43 different peptides, then incubated for 1 hour at 37°C.

6) 100 µL per well was applied of donkey anti-Sheep IgG Alkaline Phosphatase (Sigma, 1:5000 in PBS), then incubated for 1hour at 37°C.

7) 100 µL per well was applied of p-Nitrophenyl Phosphate (PNPP) substrate (Sigmafast tablets used according to the manufacturer’s instructions), then incubated for 30 minutes at 37°C.

8) The color signal was read at 405nm.

9) The absorption signals achieved from the various combinations of MABs and captured peptides were used to map the epitope of the various a-SN MABs against the various peptides.

In between each step 2-7, an automated plate washer was used to do 3 x 300 µL/well washes using PBS 0.1% Tween 20.

The epitopes identified for the tested sheep a-SN MABs are summarized in Figure 3. Wherein SNNT: N-terminal binding a-SN monoclonal antibodies, and SNCT: C-terminal binding a-SN monoclonal antibodies.

[0084] Example 4: Specificity of sheep a-SN monoclonal antibodies (towards SN versus SN and secretogranin II (SGII)).

[0085] i) SGII coated on microtiter plates:

1) Nunc Maxisorp plates were coated overnight at 2-8°C adding 100 µL per well recombinant SGII (Sino Biologicals) at 0.5 µg/mL diluted in 10 mM phosphate buffer, 0.150 M NaCl, 0.1% azide, pH 7.4 (PBS).

2) The plates were then blocked with 200 µL of milk powder (Sigma) 20 mg/mL in PBS per well for > 30 minutes at 37°C.

3) 100 µL of each of the a-SN MABs tested diluted in 10 mg/mL BSA in PBS were then added per well and incubated for 1 hour at 37°C.

4) 100 µL per well was applied of donkey anti-sheep IgG Alkaline Phosphatase (Sigma, 1:5000 in PBS), then incubated for 1 hour at 37°C.

5) 100 µL per well was applied of p-Nitrophenyl Phosphate (PNPP) substrate (Sigmafast tablets according to the manufacturer’s instructions), then incubated for 30 minutes at 37°C.

6) The color signal was read at 405 nm.

In between each step 1-5, an automated plate washer was used to do 3 x 300 µL/well washes using PBS 0.1% Tween 20.

The binding of various sheep a-SN MABs towards SGII coated plates is shown in Figure 4.

[0086] ii) Cross-reactivity towards secretogranin II (SGII), assessed using SGII in solution, not coated on microtiter plates:

1) Nunc Maxisorp plates were coated overnight at 2-8°C adding 100 µL per well SNCT:3A7 at 1 µg/mL diluted in 10mM phosphate buffer, 0.150 M NaCl, 0.1% azide, pH 7.4 (PBS).

2) The plates were then blocked with 200 µL of milk powder 20mg/mL in PBS per well for > 30 minutes at 37°C.

3) 100 µL of recombinant SGII, alternatively SN, at various concentrations diluted in 10 mg/mL BSA in PBS was then added per well and incubated for 1 hour at 37°C.

4) 100 µL per well was applied of a polyclonal rabbit a-SN antibody<(*)>-HRP conjugate (diluted 1:5000) before incubated for 1 hour at 37°C.

5) 100 µL per well was applied of TMB substrate (Sigma tablets used according to the manufacturer’s instructions), then incubated for 30 minutes at 37°C before adding 100 µL 1M H2SO4 per well as stop reagent.

6) The color signal was read at 450nm.

In between each step 1-5, an automated plate washer was used to do 3 x 300µL/well washes using PBS 0.1% Tween 20.

The results of the assessment of cross-reactivity towards SGII using free SGII in solution are presented in Figure 5. When adding non-coated/free SGII, alternatively free SN, the monoclonal C-terminal SNCT.3A7 can, together with the N-terminal binding polyclonal rabbit a-SN antibody (PAB), form a MAB II-SGII-PAB sandwich and a MAB II-SN-PAB sandwich, respectively.

<(*) >The polyclonal a-SN antibody used in this experiment had earlier been found to be SN N-terminal binding, binding towards SN’s N-terminal sequence TNEIVEEQYT. This polyclonal antibody could, as the only a-SN antibody tested, pair and form a PAB-SN-MAB II sandwich with 3A7 (contrary to any of the sheep a-SN monoclonals). When testing this polyclonal a-SN antibody in a separate experiment identical to patent experiment 3 i), the antibody was shown to also cross-reacted with SGII.

[0087] Example 5: Representative standard curves for three main immunoassay design formats.

[0088] 5i) Bead-based sandwich immunoassay for measuring secretoneurin using 2.8 µm diameter tosyl-activated paramagnetic particles:

1) Reagents were prepared as follows:

a) Preparation of microparticles coated with a-SN MAB:

Conjugation of a-SN MAB (SNCT:3B7) onto tosyl activated microparticles (Dynabeads, M280, Thermo-Fisher) was carried out using 20 µg MAB per mg paramagnetic particles (MPs). Before starting the conjugation, the MPs were washed twice in 0.1 M Borate pH 9.5, then resuspended in 1 mg/mL a-SN MAB according to the aforementioned ratio, 0.1 M Borate pH 9.5, and 3 M ammonium sulphate pH 9.0 for a final ammonium sulphate concentration of 1 M. The conjugation was left for 18 hours with rotation at 37 °C. The MPs were then incubated in 5 mg/mL BSA in 10 mM phosphate buffer, 150 mM NaCl, pH 7.4 (PBS) for 1 hour at 37 °C with rotation, before washed in 1mg/mL BSA in PBS for a total of 4 times, and then resuspended to a final concentration of 0.9 mg/mL in 1mg/mL BSA, 0.1 % NaN3 in PBS.

b) Acridine labelling of a-SN MAB:

a-SN MAB (SNNT: 2E8) was diluted in a 0.1 M carbonate buffer, pH 8.5, and 3-[9-[4-(2,5-dioxopyrrolidin-1-yl)-oxycarbonyl-2,6-dimethylphenoxy]-carbonylacridin-10-ium-10-yl]propane-1-sulfonate (NSP-DMAE-NHS) was diluted to 5 mg/mL in DMF. Conjugation of NSP-DMAE-NHS to 1 mg/mL MAB was caried out in a 20:1 NSP-DMAE-NHS to MAB molar ratio for 1 hour. The acridine labelled MAB was repeatedly purified on chromatography columns packed with Sepharose G-25 (Cytiva) until free acridine was removed. The conjugate was then diluted to 0.5µg/mL in 10 mM phosphate buffer, 150 mM NaCl, 0.5 mg/mL BSA, 0.1% Pluronic F-127 (Sigma Aldrich/Merck), pH 7.4

c) Assay buffer:

10 mM phosphate buffer, 150 mM NaCl, 0.5 mg/mL BSA, 0.1% Pluronic F-127 (Sigma Aldrich/Merck), pH 7.4

2) Reagents (1 a-c) were loaded onto an automated immunoassay instrument platform (Stratec, Germany) and used to analyze samples and calibrators according the following assay protocol: transfer 10µL of particle reagent (1 a) and 80 µL of buffer (1 c) into a reaction cup, add 10 µL of SN calibrator (crystalline SN (Genscript) dissolved in PBS buffer) or patient sample (serum, EDTA plasma or heparinized plasma) and finally 80 µL of acridine-MAB reagent (1 b). Let the reaction mixture incubate for 10 minutes at 37°C, before washing the particles three times in instrument wash buffer (tris buffered saline). The washed particles were then added 260µL of a pre-trigger solution (H2O2 in nitric acid 0.5%,) and 260µL of trigger solution (0.25 N NaOH) starting the chemiluminescence reaction. The light emitted from the calibrators and samples were recorded by the instrument and used to establish a standard curve for SN (later used to calculate the concentration of SN in unknown patient samples, please refer to patent experiment 10).

[0089] The representative standard curve for the bead-based sandwich immunoassay for measuring SN is presented in Figure 6. The x-axis shows the concentration of the SN calibrators, the y-axis the luminescence signal. Results were achieved using 2.8µm diameter paramagnetic beads functionalized with a-SN-MAB 3B7 (immobilized using epoxy activated microparticles) and NSP-DMAE labelled a-SN MAB 2E8. A viable linear calibration curve for measuring SN in unknown samples with an analytical range close to 250 pM is provided. Error bars are presented, but not observable because of the high precision analysis achieved.

[0090] 5ii) Bead-based competitive immunoassay for measuring secretoneurin based on 2.8 µm diameter paramagnetic particles coated with SN as the catching component, and acridine labelled a-SN MAB as the signaling component:

1) Reagents a-c were prepared as follows:

a) Preparation of microparticles coated with secretoneurin (SN):

Conjugation of SN onto tosylactivated microparticles (Dynabeads, M280, Thermo-Fisher) was carried out using 5 mg beads. The beads were washed twice in 1 mL 0.1M Borate pH 9.5 and resuspended in 100 µL 1 mg/mL SN, 50 µL 0.1M Borate pH 9.5 and 100 µL 3M ammonium sulphate in 0.1M Borate pH 9.0. The conjugation was left for 18 hours with rotation at 37 °C. After the incubation the paramagnetic particles were washed in 1 mL 1 mg/mL BSA in 10 mM phosphate buffer, 150 mM NaCl, pH 7.4 (PBS) for a total of 4 times, and then resuspended to a final concentration of 0.9 mg/mL in 1 mg/mL BSA, 0.1 % NaN3 in PBS. The paramagnetic particle stock concentration was 0.45 mg/mL.

b) Acridine labelling of a-SN MAB 2E8 was prepared as described in patent example 5i).

c) Assay buffer: 0.5 mg/mL BSA 0.1% Pluronic F-127 (Sigma Aldrich/Merck) in 10 mM phosphate buffer, 150 mM NaCl, pH 7.4.

2) The reagents (1 a-c) were loaded onto an automated immunoassay instrument platform (Stratec, Germany) and used to analyze SN calibrators according to the following assay protocol: 10 µL SN sample was added to a cuvette followed by 10 µL 0.45 mg/mL paramagnetic particles (1a) and 100 µL assay buffer (1c). Finally, 100 µL NSP-DMAE-MAB (1b) was added and the cuvette mix incubated for 10 minutes at 37°C before the particles were washed, pre-trigger and trigger solution was added and luminescence measured as described in patent experiment 5i).

[0091] The representative standard curve for the bead-based sandwich immunoassay for measuring SN is presented in Figure 7. The x-axis shows the final concentration of SN after 1:1 dilution with acridine labelled MAB, which had a final concentration of 0.1 µg/mL in the preincubation mix. Each dot displayed is the average of two replicates per calibrator conc. A 4-parameter logistic sigmoidal function was used to fit the calibrator response curve allowing the concentrations of SN to be determined in unknown samples.

[0092] 5iii) Bead-based filter immunoassay for measuring secretoneurin based on 2.8µm diameter non-magnetic particles coated with a-SN MAB I.

1) Reagents were prepared as follows:

a) Preparation of microparticles coated with a-SN MAB I:

Conjugation of a-SN MAB (SNCT:3B7) onto tosyl-activated microparticles (Dynabeads, M280, Thermo-Fisher) was carried out as described in Example 5 i). End particle concentration 0.9 mg/mL.

b) Assay buffer:10 mM phosphate buffer, 150 mM NaCl, 0.5 mg/mL BSA, 0.1% Pluronic F-127, pH 7.4

c) SN calibrators: Recombinant SN (Genscript) was diluted in Assay buffer (b) to the following concentrations 48, 125 and 262 pM. Assay buffer (b) was used as the zero-calibrator.

d) Preparation of HRP conjugated a-SN MAB: a-SN MAB (2E8) was HRP-labelled using an activated peroxidase labelling kit (EZ-Link, Thermo-Fisher) following the manufacturers labelling instructions. The conjugate was diluted in to 0.75 µg/mL in assay buffer (b).

e) Wash solution: 10mM Phosphate buffer pH 7.4 150mM NaCl 0.1% Tween 20.

2) An in-house filter device was made to mimic a typical flow-through point-of-care device. All separations and washings steps were performed using this device. The device was composed of two plastic layers, a top layer with 5 mm circular wholes to apply the sample, and a solid base layer. A filter disk (10 mm) and a pad (15 mm disk) was placed on top of each other in between the plastic layers before clamping the layers together to secure that all movements of liquid went through the filter and down into the pad. The filter used, SUPOR 800 WE4 membrane, as well as the porous absorption pad were both from Pall. The pad absorbed the liquids applied on top of the filter through the circular whole of the device’s top layer, allowing the particles applied to be both washed and trapped on top of the membrane.

3) The reagents (1 a-d) were mixed in a sample tube (10 µL particles (a), 1µL HRP conjugate (d), 10 µL SN calibrators (c) and 80 µL assay buffer (b)) and incubated on a shaker at room temperature for 60 minutes.

4) 100µL of the mixture was applied in the hole on top of the filter device.

5) 100µL wash solution (e) was applied in the hole on top of the filter device.

6) The filter device was dismantled, and the membrane disk placed in a vial containing 100µL TMB substrate (KEM-EN-TEC) and incubated on a shaker at room temperature for 60 minutes.

7) The substrate was then transferred to a Maxisorp plate (Nunc) and added 50 µL 0.5M H2SO4.

8) The color signal was read at 450 nm.

[0093] The representative standard curve for the non-magnetic bead-based sandwich immunoassay for measuring SN is presented in Figure 8. Using 2.8µm particles functionalized with a-SN MAB 3B7 and HRP-labelled 2E8 a viable linear calibration curve for measuring SN in unknown samples with an analytical range to 260 pM was obtained.

[0094] Example 6: Effect of increased amounts of particles in the assay demonstrated in a bead-based sandwich immunoassay design for determining secretoneurin using 2.8 µm diameter paramagnetic particles functionalized with a-SN MAB 2E8 and acridine (NSP-DMAE) labelled a-SN MAB 3B7.

1) Reagents a, b, c were prepared as described in experiment 5 ii).

The loading of the microparticles prepared (as µg a-SN MAB per mg of particles) was determined to 6.1µg/mg).

2) To demonstrate the effect of increasing the amount of microparticles used in the assay, increasing amounts of particle reagent 1a (9 µg, 18 µg and 36 µg per assay) was loaded into an automated immunoassay instrument platform (Stratec, Germany) together with reagent 1b and 1c. SN calibrators were analyzed to assess the effect of particle amount per assay. Microparticle concentrations of 0.9 mg/mL and 1.8 mg/mL were prepared from reagent 1a. Sample luminescence was measured according to variations of the following protocol: transfer 10µL of particle reagent and 80 µL of buffer (1c), alternatively 20µL of particle reagent and 70 µL of buffer) into a reaction cuvette, add 10 µL of SN calibrator, and finally 80 µL of acridine-MAB reagent (1b). Let the reaction mixture incubate for 10 minutes, before washing the particles three times in system wash buffer (tris buffered saline). To the washed particles 260µL of a pre-trigger solution (acidic H2O2 solution) and 260µL of trigger solution (NaOH) were added, starting the chemiluminescence reaction. The light emitted were recorded by the instrument and used to establish a standard curve for SN.

[0095] Results showing the analytical immunoassay signal as a function of particle amount and the linear relationship between deltaRLU and the amount of magnetic bead particles are presented in Figure 9. Curve a) is showing the analytical signal of the assay as a function of particle amount used (9-7236 µg per assay). Curve b) is showing the close to linear relationship between delta RLU (signal difference between high SN-calibrator and zero calibrator) and the amount of particles added (as “x” times 9 µg of particles per assay). Viable linear calibration curves for measuring SN in unknown samples up to 300 pM is achieved for all reaction mixtures.

[0096] Example 7: Assay performance of a particle-based immunoassay for analyzing secretoneurin.

1) Assay reagents a-c were prepared as described in patent example 5 i).

2) Reagents (a-c) were loaded onto an automated immunoassay instrument platform (Stratec, Germany) to analyze SN calibrators and unknown samples (EDTA plasma) according to the assay protocol described in patent example 5 i) step 2.

3) The following studies were performed:

a) Assay linearity

A preparation of homogenized pig brain was diluted in assay buffer to a concentration of 2300pM and labelled “High sample”. Assay buffer was labelled “Low sample”. High and low samples were mixed in different ratios to prepare 8 new samples and the 10 different samples were all analyzed using the described bead based assay for determining the SN concentration. The theoretical SN concentration of each sample was calculated based on the measured concentration of the high and the low sample, and the measured SN concentration was then plotted against the theoretical value for each sample.

b) Correlation with SN-ELISA

40 serum samples previously analyzed using a commercially available SN ELISA (CardiNor AS, Norway) were reanalyzed using the described bead based immunoassay for measuring secretoneurin. Measured SN concentration of patient samples were then plotted against the corresponding concentration of SN measured using the ELISA.

c) Imprecision of SN measurements

Two samples were used: one sample with low SN concentration (20pM) and one with high SN concentration (100pM). These samples were analyzed in 20 replicates using the described particle based assay. Relative imprecision based on the measured SN concentrations was estimated for each of the two samples.

[0097] Results of the immunoassay linearity and the correlation with a commercially available SN-ELISA are presented in Figure 10 a) and b), respectively. Curve a) presents assay linearity. The test result for each of the 10 samples analyzed is presented as the mean result of the seven replicates. The bead-based sandwich immunoassay described for measuring secretoneurin is linear up to an SN concentration of at least 2300 pM. Curve b) shows the correlation with SN-ELISA. SN results achieved using the described particle-based method compared with the corresponding results achieved using a commercially available SN ELISA. Linear correlation, y = 0.99x – 2, R2= 0.89.

[0098] Table 4 presents the analysis results of two different patient samples.

Table 4. Table listing the results after analyzing two different patient samples (EDTA plasma). One sample with low SN concentration (25 pM), and one sample with high SN concentration (100pM) respectively, in 20 replicates using the bead-based method described. Calculated average and imprecision (%CV) are shown.

[0099] Table 5 summarizes key parameters used to assess the performance and characteristics of the preferred embodiment of the present invention.

Table 5. Summary of results showing performance and characteristics of the particlebased method of the invention for measuring SN (using paramagnetic particles coated with MAB I and acridine labelled MAB II) compared to a commercially available SN ELISA.

<(*) >Dependent upon no. of samples analyzed

<(**) >Imprecision (%CV) of instrument platform used: ≃2%

<(***)>Trained operator

Claims

1. An immunoassay for determining secretoneurin peptide concentration in a fluid sample, the immunoassay comprising a mixture of

at least one monoclonal antibody with binding specificity exclusively towards secretoneurin and without cross-reactivity towards the precursor protein secretogranin II (SGII, chromogranin C) (a-SN MAB I);

a signaling component, wherein the signaling component comprises a label; at least one assay buffer;

2. The immunoassay according to claim 1, wherein

an antibody or antigen-binding fragment thereof cross-blocks the a-SN MAB I from binding to secretoneurin; and

an antibody or antigen-binding fragment thereof cross-blocks the signaling component from binding to secretoneurin, wherein the signaling component comprises a sheep monoclonal a-SN antibody with binding specificity towards the N-terminal region of SN comprising CDRs of any of reference id: a-f, being amino acid sequences RSSLTN, YMSSSGRTG, YDLVY, SGSSNNVGYGNYVN, SVTSRAS, TSYDISGNNI.

3. The immunoassay according to any of the claims 1 to 2, wherein the immunoassay is a sandwich immunoassay, wherein the mixture comprises

the catching component comprises the at least one a-SN MAB I;

the signaling component comprises a monoclonal SN antibody with binding affinity towards the C- or N-terminal region of SN (SN MAB II).

4. The immunoassay according to any of the claims 1 to 3, wherein the catching component comprises the at least one a-SN MAB I, wherein the a-SN MAB I has binding specificity towards the C-terminal region of SN and the signaling component comprises a monoclonal SN antibody with binding affinity towards the N-terminal region of SN (SN MAB II).

5. The immunoassay according to any one of claims 1 to 3, wherein the catching component comprises a monoclonal SN antibody with binding affinity towards the N terminal region of SN (SN MAB II) and the signaling component comprises the at least one a-SN MAB I.

6. The immunoassay according to claim 1, wherein the immunoassay is a competitive assay, wherein the mixture comprises

the catching component comprises the at least one a-SN MAB I;

the signaling component comprises an SN analog.

7. The immunoassay according to claim 1, wherein the immunoassay is a competitive assay, wherein the mixture comprises

the catching component comprises a SN analog;

the signaling component comprises the at least one a-SN MAB I.

8. The immunoassay according to any one of claim 1-7, wherein the at least one a-SN MAB I is from sheep.

9. The immunoassay according to any one of claims 1-8, wherein the solid phase comprises bead particles in the form of either superparamagnetic bead particles or non-magnetic bead particles, wherein the bead particles comprise a surface and wherein the bead particle size is in the range between 0.5-5 μm for superparamagnetic beads and the bead particle size is in the range between 100-5000 nm for non-magnetic bead particles.

10. The immunoassay according to any of claims 1-9, wherein the solid phase comprises superparamagnetic bead particles with a surface adapted to enable loading of > 1 μg of a a-SN MAB I, a SN MAB II, or an SN analog per mg bead particle.

11. The immunoassay according to any one of claims 1-10, wherein the catching component is conjugated to the surface of the solid phase through covalent linkage of a functional group of the catching component to a functional group of the solid phase.

12. The immunoassay according to any of claims 1-11, wherein the label of the signaling component is linked to the at least one a-SN MAB I, to a monoclonal SN antibody with binding affinity towards the N-terminal region of SN (SN MAB II), or to an SN analog by reacting a functional group of the a-SN MAB I, the SN MAB II, or the SN analog with a functional group of the label.

13. The immunoassay according to any of the preceding claims, wherein the label of the signaling component is selected from the group comprising enzymes, fluorescent substances, luminescent substances, and particles.

14. A method for determining SN peptide concentration in a fluid sample from a subject, using the immunoassay according to any of the claims 1 to 13.

15. An immunoassay method for determining secretoneurin (SN) peptide concentration in a fluid sample, wherein the method is a sandwich immunoassay method comprising a sequence of steps including to:

a catching component comprising a monoclonal antibody (MAB I) with binding specificity exclusively towards secretoneurin (SN) and without cross-reactivity towards the precursor protein secretogranin II (SGII, chromogranin C), a solid phase, wherein the MAB I is immobilized onto;

at least one assay buffer,

(4) Wash the retained MAB I-SN-MAB II sandwich with wash buffer,

(5) Optionally, activate the label on the MAB II.

16. An immunoassay method for determining secretoneurin (SN) peptide concentration in a fluid sample, wherein the immunoassay method is a competitive immunoassay method comprising a sequence of steps including to:

(1) Mix a fluid sample from a subject with immunoassay components, the immunoassay components comprising a mixture of:

a catching component comprising a monoclonal antibody (MAB I) with binding specificity exclusively towards secretoneurin (SN) and without cross-reactivity towards the precursor protein secretogranin II (SGII, chromogranin C) or towards a secretoneurin (SN) analog,

a solid phase wherein the catching component is immobilized onto;

a signaling component, wherein the signaling component comprises a label attached to a secretoneurin (SN) analog or a label attached to a MAB I; at least one assay buffer,

(5) Optionally, activate the label on the signaling component.