A method for the specific determination of proenkephalin fragment 119-159

Abstract

Subject matter of the present invention is the use of at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (SEQ ID No. 6) for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid. Further subject matter of the present invention is a kit for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) comprising at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid. Further subject matter of the present invention is also a method for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid, the method comprising: providing a sample of bodily fluid of a subject and determining the level of proEnk fragment 119-159 in said sample of bodily fluid, using at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

Description

[0001] A method for the specific determination of Proenkephalin fragment 119-159

[0002] Subject matter of the present invention is the use of at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (SEQ ID No. 6) for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid.

[0003] Further subject matter of the present invention is a kit for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) comprising at least three binders binding to three different nonoverlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid.

[0004] Further subject matter of the present invention is also a method for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid, the method comprising: providing a sample of bodily fluid of a subject, determining the level of proEnk fragment 119-159 in said sample of bodily fluid, using at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0005] State of the Art

[0006] Proenkephalin A is a precursor of the enkephalin family of endogenous opioids. It is a prohormone that is proteolytically processed to form several active pentapeptides like methionine-enkephalin (Met-Enk) and leucine-enkephalin (Leu-Enk) together with several other peptide fragments (enkelytin and C-terminal extended Met-Enk peptides). In addition to mature enkephalins, other peptides are produced, one of which is a stable proenkephalin peptide 119-159 (proEnk fragment 119-159). This peptide fragment levels in plasma / serum could serve as a surrogate measurement of systemic enkephalin synthesis, because proenkephalin is the predominant source of mature enkephalins. (Ernst et al. , 2006. Peptides 27: 1835-1840}. Enkephalins are widely secreted to act on locally expressed opioid receptors, specifically the 5 opioid receptors. These opioid receptors are also widely expressed, with the highest density found in the kidney (Denning et al. 2008. Peptides 29 (1): 83-921}. After receptor binding, the biological effects of enkephalins include nociception, anesthetics, and cardiovascular regulation (Holaday 1983. Annu. Rev. Pharmacol. Toxicol. 23: 541-594}. These 5 opioid agonists stimulate natriuresis and diuresis (Sezen et al. 1998. J. Pharmacol. Exp. Ther. 287 (1): 238-245}. While several studies have demonstrated that elevated concentrations are associated with adverse outcomes, the association has in general been proportional to the change in renal function. Indeed, increased concentrations are associated with decreased renal function in several populations including sepsis (Marino et al. 2015. J Nephrol 28: 717 724), heart failure (Ng etal. 2017. J. Am. Coll. Cardiol. 69 (1): 56-69; Matsue etal. 2017. J. Card. Fail. 23 (3): 231-239), cardiac surgery (Shah et al. 2015. Clin. Nephrol. 83 (1): 29-35), and myocardial infarction (Ng et al. 2014. J. Am. Coll. Cardiol. 63 (3) (2014) 280-289). proEnk fragment 119-159 strongly correlates with the kidney function and measured GFR (Beunders et al. 202054(3): 308-314). Therefore, it was proposed as a biomarker for assessing kidney function in critically ill patients (Beunders et al. 2017. Appl Lab Med 2(3): 400-412; Donato et al. 2018. Clin Biochem 58: 72-77; Beunders et al. 2020 54(3): 308-314; Khorashadi et al. 2020. Nephron 144(12): 655-661). Moreover, low fasting plasma concentration of the proEnk fragment 119-159 is associated with an increased risk of future breast cancer in middle-aged and postmenopausal women (Melander et al. 2015. J Clin Oncol 33(24): 2632-2638) .

[0007] Immunoassays for the determination of proEnk fragment 119-159 have been described recently by Ernst et al. (Ernst et al. 2006. Peptides 27: 1835-1840) and by Donato et al. (Donato et al. 2018. Clin Biochem 58: 72-77). Ernst et al. described an immunoassay using a monoclonal anti- proEnk 121-134 antibody as solid phase antibody and a polyclonal (sheep) anti-Enk 139-155 antibody as tracer antibody, respectively. Donato et al. described an immunoassay using two monoclonal antibodies directed against amino acids 129-144 of proEnk (antibody against the mid- regional part) and amino acids 152-159 (antibody against the very C-terminal part). Both assays do not detect proEnk fragment 119-159 specifically by using both, antibodies against the very-C- terminal and very-N-terminal part of the molecule. On the contrary, attempts to measure proEnk fragment 119-159 specifically by using antibodies that recognize the very-C-terminal and very-N- terminal part of proEnk fragment 119-159 did not result in any signal generation at all ( W02014 / 053501 and Example 1 Table 4).

[0008] It was therefore the task to provide an assay that specifically detects the proEnk fragment 119-159 by using antibodies against the very N-terminal and C-terminal part of the molecule. Surprisingly, if an immunoassay was performed using three antibodies binding to three different nonoverlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (very-N-terminal part, very-C-terminal part and mid-regional part, respectively) there was not only a signal generated, but the signal was significantly higher compared to immunoassay setups for proEnk 119-159 described in the art. The present invention describes a sensitive method for the specific detection of the proEnk fragment 119-159. Detailed Description

[0009] Subject matter of the present invention is the use of at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (SEQ ID No. 6) for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid.

[0010] Further subject matter of the present invention is a kit for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) comprising at least three binders binding to three different nonoverlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid.

[0011] Further subject matter of the present invention is also a method for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid, the method comprising: providing a sample of bodily fluid of a subject, determining the level of proEnk fragment 119-159 in said sample of bodily fluid, using at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0012] Surprisingly, it has been shown that the use of three binders binding to three different nonoverlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (very-N-terminal part, very-C-terminal part and mid-regional part, respectively) resulted in a sensitive and specific detection of the proEnk fragment 119-159.

[0013] The terms Pro-Enkephalin, proenkephalin, proEnk and PENK are used synonymously throughout the specification.

[0014] Pro-Enkephalin has the following sequence:

[0015] SEQ ID NO. 1 (Pro-Enkephalin 1-243)

[0016] ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTL RENSKPEESHLLAKRYGGFMKRYGGFMKKMDELYPMEPEEEANGSEILAKRYGGFMK KD AEEDD SL ANS SDLLKELLETGDNRERSHHQDGSDNEEE VSKRYGGFMRGLKRSPQL EDEAKELQKRYGGFMRRVGRPEWWMDYQKRYGGFLKRFAEALPSDEEGESYSKEVPE MEKRYGGF MRF The following fragments or partial peptides of Pro-Enkephalin are used throughout the specification:

[0017] SEQ ID NO. 2 (Peptide-DD14)

[0018] DAEEDDSLANSSD

[0019] SEQ ID NO. 3 (Peptide SE-17)

[0020] S SDLLKELLETGDNRE

[0021] SEQ ID NO. 4 (Peptide TE-18)

[0022] TGDNRERSHHQDGSDNE

[0023] SEQ ID NO. 5 (Peptide SS-8)

[0024] SDNEEEVS

[0025] SEQ ID NO 6: (Pro-Enkephalin 119-159, Mid-regional Pro-Enkephalin-fragment, MR-PENK) DAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVS

[0026] An epitope, also known as antigenic determinant, is the part of an antigen (e.g., peptide or protein) that is recognized by the immune system, specifically by antibodies. For example, the epitope is the specific piece of the antigen to which an antibody bind. The part of an antibody that binds to the epitope is called a paratope. The epitopes of protein antigens are divided into two categories: conformational epitopes and linear epitopes, based on their structure and interaction with the paratope.

[0027] A linear or a sequential epitope is an epitope that is recognized by antibodies by its linear sequence of amino acids, or primary structure and is formed by the 3-D conformation adopted by the interaction of contiguous amino acid residues. Conformational and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the involved epitope residues and the shape or tertiary structure of other segments of the antigen. In contrast, a conformational epitope is formed by the 3-D conformation adopted by the interaction of sequentially discontinuous but close together in three-dimensional space amino acid residues.

[0028] In one embodiment the three different non-overlapping epitopes of proEnk fragment 119-159 are within the N-terminal region that is amino acid 119 to 133 (SEQ ID No. 2), within the mid-regional region that is amino acid 129 to 155 (SEQ ID No. 7) and within the C-terminal region that is amino acid 152 to 159 (SEQ ID No. 5), wherein each of said epitopes comprises at least 4 or 5 amino acids.

[0029] In one embodiment said determination of the level of proEnk fragment 119-159 is a quantitative or qualitative determination.

[0030] In another embodiment said determination is a specific determination of proEnk fragment 119- 159, wherein said binder binding to the N-terminal region recognizes and binds to the N-terminal end (amino acid 119, which is aspartic acid) of proEnk fragment 119-159 and wherein said binder binding to the C-terminal region recognizes and binds to the C-terminal end (amino acid 159, which is serine) of proEnk fragment 119-159. Specific means that proEnk fragment 119-159 is specifically detected, but no elongated or truncated forms of the molecule.

[0031] In another embodiment said binder binding to the N-terminal region recognizes and binds to the N-terminal end (binds to amino acid 119, which is aspartic acid) of proEnk fragment 119-159 and wherein said binder binding to the C-terminal region recognizes and binds to the C-terminal end (binds to amino acid 159, which is serine) of proEnk fragment 119-159.

[0032] In the context of the present invention, “binder molecules” are molecules which may be used to bind target molecules or molecules of interest, i.e. analytes (i.e. in the context of the present invention proEnk fragment 119-159), from a bodily fluid sample. Binder molecules must thus be shaped adequately, both spatially and in terms of surface features, such as surface charge, hydrophobicity, hydrophilicity, presence or absence of lewis donors and / or acceptors, to specifically bind the target molecules or molecules of interest. Hereby, the binding may for instance be mediated by ionic, van-der-Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bond interactions or a combination of two or more of the aforementioned interactions between the binder molecules and the target molecules or molecules of interest. In the context of the present invention, binder molecules may for instance be selected from the group comprising a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide or a glycoprotein. Preferably, the binder molecules are antibodies, including fragments thereof with sufficient affinity to a target or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and / or biochemically modified derivatives of said antibodies or fragments derived from the variant chain with a length of at least 12 amino acids thereof.

[0033] In one embodiment of the invention said binder is selected from the group comprising an antibody, an antibody fragment, aptamer or a non-Ig-scaffold binding to proEnk fragment 119-159.

[0034] According to the invention the binder to proEnk fragment 119-159 is selected from the group consisting of antibodies e.g. IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F-variable domain of heavy and / or light chain as e.g. chemically coupled antibodies (fragment antigen binding) including but not limited to Fab-fragments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g. Fab- V5Sx2; bivalent Fab (mini-antibody) dimerized with the CEE domain; bivalent Fab or multivalent Fab, e.g. formed via multimerization with the aid of a heterologous domain, e.g. via dimerization of dHLX domains, e.g. Fab-dHLX-FSx2; F(ab‘)2-fragments, scFv-fragments, multimerized multivalent or / and multi-specific scFv-fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T-cell engager), trifunctional antibodies, polyvalent antibodies, e.g. from a different class than G; single-domain antibodies, e.g. nanobodies derived from camelid or fish immunoglobulins. In a specific embodiment of the invention said binders to proEnk 119-159 are full-length immunoglobulin G (IgG).

[0035] Said binder may be a “capture binder”, wherein said capture binder is unlabelled and immobilized to a solid phase.

[0036] The term “antibody” generally comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single-chain-antibodies” (Bird et al. 1988), chimeric, humanized, in particular CDR-grafted antibodies, and dia- or tetrabodies (Holliger et al. 1993). Also comprised are immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample. In this context the term “specific binding” refers to antibodies raised against the molecule of interest or a fragment thereof. An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least preferably 50-fold higher, more preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to make antibodies and to select antibodies with a given specificity.

[0037] An antibody or fragment according to the present invention is a protein including one or more polypeptides substantially encoded by immunoglobulin genes that specifically binds an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgGl, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 KDa or 214 amino acids in length. Full-length immunoglobulin heavy chains are generally about 50 KDa or 446 amino acids in length. Light chains are encoded by a variable region gene at the NFE-terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus. Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0038] The basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al. 1987. Eur. J. Immunol. 17: 105; Huston et al. 1988. Proc. Natl. Acad. Sci. U.S.A., 85: 5879-5883; Bird et al. 1988. Science 242: 423-426; Hood et al. 1984, Immunology, Benjamin, N.Y., 2nd ed.; Hunkapiller and Hood 1986. Nature 323:15-16}. An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al. 1983, U.S. Department of Health and Human Services}. As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen. An immune complex is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody or human antibody, or functional antibody fragment, specifically bound to the antigen.

[0039] Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent No. 5,807,715.

[0040] A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor" and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDR’s. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Patent No. 5,585,089). A human antibody is an antibody wherein the light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be produced by phage display methods (see, e.g., WO91 / 17271; WQ92 / 001047; WO92 / 2Q791) or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (for example, see WO93 / 12227; WO 91 / 10741}.

[0041] Humanization of murine antibodies may be conducted according to the following procedure: For humanization of an antibody of murine origin the antibody sequence is analyzed for the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modelling (Almagro andFransson 2008. Humanization of antibodies. Front Biosci. 13:1619-33}.

[0042] Methods for obtaining monoclonal antibodies

[0043] In all of the following embodiments, the term monoclonal antibody is meant to include monoclonal antibodies, as well as fragments of monoclonal antibodies, such as the ones detailed herein, more particularly monoclonal antibodies.

[0044] Hybridoma

[0045] In a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain a multitude of hybridomas, ii) isolating from said multitude of hybridomas a hybridoma producing a desired monoclonal antibody.

[0046] In certain embodiments, the antibody according to the present invention is a monoclonal antibody obtainable by isolating from a multitude of hybridomas a hybridoma producing a desired monoclonal antibody, wherein said multitude of hybridomas were produced by fusing antibodysecreting cells from an animal previously immunized with an antigen with myeloma cells to obtain multitude of hybridomas.

[0047] A desired monoclonal antibody is in particular a monoclonal antibody binding the antigen, in particular with a binding affinity of at least 107M’1, preferred 108M’1, more preferred affinity is greater than 109M’1, most preferred greater than IO10M’1.

[0048] In certain embodiments of the method for obtaining an antibody, in step i) the animal is a mammal, particularly a rabbit, a mouse or a rat, more particularly a mouse, more particularly a Balb / c mouse. In certain embodiments of the method for obtaining an antibody, in step i) the antibody-secreting cell is a splenocyte, more particularly an activated B-cell.

[0049] In certain embodiments of the method for obtaining an antibody, in step i) fusing involves the use of polyethylene glycol.

[0050] In certain embodiments of the method for obtaining an antibody, in step i) the myeloma is derived from a mammal, in certain embodiments from the same species of mammal from which the multitude of antibody-secreting cells is obtained. In certain specific embodiments of the method for obtaining an antibody, in step i) the myeloma cells are of the cell line SP2 / 0.

[0051] In certain embodiments of the method for obtaining an antibody, said fusing in step i) comprises PEG-assisted fusion, Sendai virus-assisted fusion or electric current-assisted fusion.

[0052] In certain embodiments of the method for obtaining an antibody, said isolating in step ii) comprises performing an antibody capture assay, an antigen capture assay, and / or a functional screen.

[0053] In certain embodiments of the method for obtaining an antibody, in step ii) isolating the hybridoma producing a desired monoclonal antibody may involve cloning and re-cloning the hybridomas using the limiting-dilution technique.

[0054] In one embodiment, said antigen capture assay comprises: a) binding the produced antibodies to a substrate, particularly a solid substrate, b) allowing antigen to bind to said antibodies, c) removing unbound antigen by washing, d) detecting bound antigen; or said antigen capture assay comprises: a) allowing an antigen to bind the produced antibodies to form an antibody-antigen complex, b) binding said antibody-antigen complex to a substrate, particularly a solid substrate, c) removing unbound antigen by washing, d) detecting bound antigen. In one embodiment, said isolating of step ii) comprises performing an enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, cell staining, immunoprecipitation, and / or a western blot.

[0055] In one embodiment, said detecting of the antibody or the antigen is accomplished with an immunoassay.

[0056] In one embodiment, the animal is a transgenic animal, in particular a transgenic mouse (wherein in particular the mouse immunoglobulin (Ig) gene loci have been replaced with human loci within the transgenic animal genome), such as HuMabMouse or XenoMouse.

[0057] In one embodiment, the antigen comprises a peptide as described herein in Table 2, which in certain embodiments (in particular for immunization) may be conjugated to a protein, particularly a serum protein, more particularly a serum albumin, more particularly BSA.

[0058] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) fusing splenocytes cells from a Balb / c mouse previously immunized with a peptide as described herein in Table 2 with SP2 / 0 myeloma cells using polyethylene glycol, to obtain a multitude of hybridomas, ii) isolating from said multitude of hybridomas a hybridoma producing a desired monoclonal antibody; more preferably, the method comprises:

[0059] 1) growing hybridomas for a first period (in particular 2 weeks) in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement]

[0060] 2) followed replacing HAT medium with HT Medium for a multitude of passages (in particular 3)

[0061] 3) followed by returning to the normal cell culture medium for a second time period, in particular until the end of three weeks after fusion

[0062] 4) primary screening of cell culture supernatants for antigen-specific IgG antibodies

[0063] 5) propagating microcultures of cells that tested positive in 4)

[0064] 6) retesting cell culture supernatants of microcultures for antigen-specific IgG antibodies 7) cloning and re-cloning cultures that tested positive in 6), using the limiting-dilution technique

[0065] 8) optionally determining the isotypes of clones obtained from 7)

[0066] 9) optionally purifying antibodies via Protein A

[0067] Phage Display

[0068] In a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) isolating at least one antibody having affinity to an antigen from an antibody gene library; ii) generating at least one cell strain expressing said at least one antibody; iii) isolating the at least one antibody from a culture of the at least one cell strain obtained in step ii).

[0069] An antibody having affinity to an antigen is in particular an antibody with a binding affinity of at least 107M’1, preferred 108M’1, more preferred affinity is greater than 109M’1, most preferred greater than IO10M'1.

[0070] In a certain embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by isolating at least one antibody from a culture derived from at least one cell strain which expressed at least one antibody having affinity to an antigen from an antibody gene library.

[0071] In one embodiment, the antigen comprises a peptide as described herein in Table 2, which in certain embodiments may be bound to a solid phase.

[0072] In certain embodiments of the method for obtaining an antibody, in step i) the antibody gene library is a naive antibody gene library, particularly a human naive antibody gene library, more particularly in said library the antibodies are presented via phage display, i.e. on phages comprising a nucleotide sequence encoding for such respective antibody; more particularly the library HAL 7, HAL 8, or HAL 9, more particularly a library comprising the human naive antibody gene libraries HAL7 / 8. In certain embodiments of the method for obtaining an antibody, in step i) screening comprises the use of an antigen, particularly an antigen containing a tag, more particularly a biotin tag, linked thereto via two different spacers. In particular embodiments, such panning strategy includes a mix of panning rounds with non-specifically bound antigen and antigen bound specifically via the tag, in the case of a biotin tag, bound to streptavidin. In this way, the background of non-specific binders may be minimized.

[0073] In certain embodiments of the method for obtaining an antibody, in step i), in embodiments wherein the library is a phage display library, the antibody is isolated by isolating a phage presenting said antibody (and comprising a nucleotide sequence encoding for the antibody).

[0074] In certain embodiments of the method for obtaining an antibody, in step ii) said cell strain is generated via introduction of a nucleotide sequence encoding for the antibody), in embodiments wherein the library in step i) is a phage display library, the isolated phage from step i) may be used to produce a bacterial strain, e.g. an E. coli strain, expressing the antibody.

[0075] In certain embodiments of the method for obtaining an antibody, in step iv); in embodiments wherein the library in step i) is a phage display library and wherein a bacterial strain is produced in step ii), antibody may be isolated from the supernatant of the culture.

[0076] It is understood that, as used in describing the methods for obtaining an antibody, the term “one antibody” in the expression “at least one antibody” in particular may include more than one antibody molecule of antibodies having the same amino acid sequence. This understanding applies, mutatis mutandis, to the term “one cell strain”.

[0077] In certain embodiments of the method for obtaining an antibody, more than one antibody (referring to a multitude of antibodies having distinct amino acid sequences, respectively) is isolated in step i) and accordingly more than one cell strain is generated in step ii). Such method may involve the selection of clones that are positive for binding to the antigen, e.g. via a binding assay, e.g. an ELISA assay involving the antigen, and cells positive for binding to the antigen may be isolated to produce monoclonal cell strains.

[0078] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) isolating at least one antibody having affinity to an antigen from an antibody gene library comprising the human naive antibody gene libraries HAL7 / 8, by eluting phages carrying said antibody from the library; ii) generating at least one E. coli cell strain expressing said at least one antibody; iii) isolating the at least one antibody from the supernantant a culture of the at least one E. coli cell strain obtained in step ii).

[0079] In a further aspect, an antibody fragment according to the present invention is produced by a method in volving enzymatic digestion of an antibody. In certain embodiments, this method produces e.g. Fab or F(ab)2 antibody fragments. In certain embodiments, this method involves digestion with pepsin or papain, which are optionally immobilized on a surface.

[0080] In certain embodiments, antibodies may be humanized by CDR-grafting, in particular by a process involving the steps: extracting RNA from hybridomas expressing an antibody of interest (e.g. obtained by a method as described herein); amplifying said extracted RNA via RT-PCR, in particular with primer sets specific for the heavy and light chains of the antibody of interest, to obtain to obtain a DNA product; further amplifying said DNA product via PCR, in particular using semi-nested primer sets specific for antibody variable regions; determining the sequence of the DNA product; aligning said sequence with homologous human framework sequences to determine a humanized sequence for the variable heavy chain and the variable light chain sequences (of the desired antibody).

[0081] In certain embodiments, antibodies may be humanized by aligning the sequence of a DNA product that was obtained by amplifying RNA extracted from hybridomas expressing an antibody of interest via RT-PCR, in particular with primer sets specific for the heavy and light chains of the antibody of interest and further amplifying the DNA obtained therefrom via PCR, in particular using semi-nested primer sets specific for antibody variable regions, with homologous human framework sequences to determine a humanized sequence for the variable heavy chain and the variable light chain sequences (of the desired antibody).

[0082] In certain embodiments, antibodies may be humanized by determining the complementary determining regions (CDR), which may be accomplished by analysing the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen;

[0083] - transplanting said CDR sequences into a human framework region.

[0084] In certain embodiments, antibodies may be humanized by transplanting CDR sequences, which may preferably have been determined by analysing the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen, into a human framework region.

[0085] In certain embodiments variations in the amino acid sequence of the CDRs or FRs may be introduced to maintain structural interactions with the antigen (which may otherwise be abolished by introducing the human FR sequences), for instance by a random approach using phage display libraries or via directed approach guided by molecular modelling.

[0086] The DNA sequences encoding for antibodies determined as detailed herein can be transferred by known genetic engineering techniques into cells and used for production of the antibody.

[0087] Producing antibodies

[0088] In a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by a method comprising: culturing a cell strain comprising a nucleotide sequence encoding for the antibody; isolating the antibody from said culture.

[0089] In a further certain aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by isolating the antibody from a culture of a cell strain comprising a nucleotide sequence encoding for said antibody.

[0090] In certain embodiments of said method, the cell strain is produced as described herein above and may comprise bacterial cells, such as gram-negative bacteria, e.g. E. coli, Proteus mirabilis. or Pseudomonas putidas, gram-positive bacteria, e.g. Bacillus brevis, Bacillus sublilis. Bacillus megaterium, Lactobacilli such as Lactobacillus zeae / casei or Lactobacillus paracasei, or Streptomyces, such as Streptomyces lividans eucariotic cells such as yeast, e.g. Pichia pasloris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe.

[0091] Schwanniomyces occidentalis, Kluyveromyces lactis or Yarrowia lipolytica, fungi, such as filamentous fungi, e.g. of the genus Trichoderma of Aspergillus, such as A. niger (e.g. subgenus A. awamori) and Aspergillus oryzae, Trichoderma reesei, Chrysosporium, such as C. hicknow ense protozoae, such as Leishmania, e.g. L. larenlolae: insect cells, such as insect cells transfected a Baculovirus, e.g. AcNPV, such as insect cell lines from Spodoptera frugiperda, e.g. Sf-9 or Sf-21, Drosophila melanogasler. e.g. DS2, or Trichopulsia ni. e.g. High Five cells (BTI- TN-5B1-4); mammalian cells such as hamster, e.g. Chinese hamster ovary such as K1-, DukX B11-, DG44, Lecl3, or BHK, mouse, e.g. mouse myeloma such as NSO, Homo sapiens, e.g. Per.C6, AGE1.HN, HEK293.

[0092] In certain embodiments of said method, the cells may be hybridoma cells, e.g. as described herein.

[0093] In certain embodiments of said method, culturing may take place in a static suspension culture, an agitated suspension culture, a membrane-based culture, a matrix-based culture or a high cell density bioreactor; a vessel for such culturing may be selected from the group comprising a T- flask, a roller culture, a spinner culture, a stirred tank bioreactor, an airlift bioreactor, a static membrane-based or matrix-based culture system, a suspension bioreactor, a fluidized bed bioreactor, a ceramic bioreactor, a perfusion system, a hollow fiber bioreactor.

[0094] In certain embodiments of said method, the cells may be immobilized on a matrix.

[0095] A high cell density bioreactor is in particular a culture system capable of generating cell densities greater than 108cells / ml.

[0096] In a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by a method comprising: generating a transgenic plant or animal comprising a nucleotide sequence encoding for the antibody; isolating the antibody from said plant or animal or a secretion or product of said plant or animal.

[0097] In a certain further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by isolating the antibody from a transgenic plant or transgenic animal or a secretion or product of a transgenic plant or transgenic animal having a nucleotide sequence encoding for the antibody.

[0098] Said animal may e.g., be selected from a chicken, a mouse, a rat, a rabbit, a cow, a goat, a sheep, a pig; said secretion or product may e.g. be milk or an egg. Said plant may e.g. be selected from tobacco (N. tabacum or A benlhamiana . duckweed (Lemna minor). Chlamydomonas reinhardlii. rice, Arabidopsis thaliana. alfalfa (Medicago saliva), lettuce, maize. The antibodies can in certain embodiments be isolated by physicochemical fractionation, e.g. size exclusion chromatography, precipitation, e.g. using ammonium sulphate, ion exchange chromatography, immobilized metal chelate chromatography gel filtration, zone electrophoresis; based on their classification e.g. binding to bacterial proteins A, G, or L, jacalin; antigen-specific affinity purification via immobilized ligands / antigens; if necessary, low molecular weight components can be removed by methods like dialysis, desalting, and diafiltration.

[0099] In some embodiments the antibody is encoded by a nucleotide sequence where the nucleotide sequence is a reverse transcription of an amino acid sequence from an antibody produced by one of the processes described herein.

[0100] Binder that may be used for determining the level of proEnk fragment 119-159 exhibit an affinity constant to proEnk fragment 119-159 of at least 107M’1, preferred 108M’1, preferred affinity constant is greater than 109M’1, most preferred greater than IO10M’1. A person skilled in the art knows that it may be considered to compensate lower affinity by applying a higher dose of compounds and this measure would not lead out-of-the-scope of the invention. Binding affinity may be determined using the Biacore method, offered as service analysis e.g. at Biaffin, Kassel, Germany (http: / / www.biaffm.com / de / ).

[0101] In addition to antibodies other biopolymer scaffolds are well known in the art to complex a target molecule and have been used for the generation of highly target specific biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins. Non-Ig scaffolds may be protein scaffolds and may be used as antibody mimics as they are capable to bind to ligands or antigens. Non-Ig scaffolds may be selected from the group comprising tetranectin-based non-Ig scaffolds (e.g. described in US 2010 / 0028995), fibronectin scaffolds (e.g. described in EP 1266 025; lipocalin- based scaffolds (e.g. described in WO 2011 / 154420); ubiquitin scaffolds (e.g. described in WO 2011 / 073214), transferring scaffolds (e.g. described in US 2004 / 0023334), protein A scaffolds (e.g. described in EP 2231860), ankyrin repeat based scaffolds (e.g. described in WO 2010 / 060748), microproteins preferably microproteins forming a cystine knot) scaffolds (e.g. described in EP 2314308), Fyn SH3 domain based scaffolds (e.g. described in WO 2011 / 023685) EGFR-A-domain based scaffolds (e.g. described in WO 2005 / 040229) and Kunitz domain based scaffolds (e.g. described in EP 1941867). In a specific embodiment the level of proEnk fragment 119-159 is determined with an immunoassay using binders, especially antibodies or fragments of antibodies binding to proEnk fragment 119-159. An immunoassay that may be useful for determining the level of proEnk fragment 119-159 may comprise the steps as outlined in Example 2.

[0102] In another specific embodiment the level of proEnk fragment 119-159 is determined with an assay using binders selected from the group comprising aptamers, non-Ig scaffolds as described in greater detail above and binding to proEnk fragment 119-159.

[0103] Subject matter of the present invention is the use of at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (SEQ ID No. 6) for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid.

[0104] Subject matter of the invention is further an assay using at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (SEQ ID No. 6) for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid.

[0105] The assays can be homogenous or heterogeneous assays, competitive and non-competitive assays. In a “classical” sandwich assay, which is a non-competitive immunoassay, the molecule to be detected and / or quantified is bound to a first antibody and to a second antibody. The first antibody may be bound to a solid phase, e.g. a bead, a surface of a well or other container, a chip or a strip, and the second antibody is an antibody which is labeled, e.g. with a dye, with a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedures involved with “sandwich assays” are well-established and known to the skilled person.

[0106] Subject matter of the invention is further a sandwich immunoassay using at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (SEQ ID No. 6) for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid.

[0107] In one embodiment at least two binders directed to two different non-overlapping regions of proEnk fragment 119-159 are capture binders and at least one binder directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 is labelled for detection. In another embodiment at least two binders directed to two different non-overlapping regions of proEnk fragment 119-159 are labelled for detection and at least one binder directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 is a capture binder. In yet another embodiment at least one binder directed to one region of proEnk fragment 119-159 is a capture binder, at least one binder directed to a second region of proEnk fragment 119-159 is labelled for detection and at least one binder directed to a third region of proEnk 119-159 is not labelled for detection and is not a capture binder, wherein the first, second and third region of proEnk 119-159 do not overlap. An overview of different combinations of binders directed to the N-terminal region that is amino acid 119 to 133 (SEQ ID No. 2), directed to the mid-regional region that is amino acid 129 to 155 (SEQ ID No. 7) and directed to the C-terminal region that is amino acid 152 to 159 (SEQ ID No. 5), with epitopes that do not overlap is summarized in table 1.

[0108] Table 1 : Overview of different combinations of binders of proEnk fragment 119-159

[0109] As mentioned herein, an “assay” or “diagnostic assay” can be of any type applied in the field of diagnostics. Such an assay may be based on the binding of an analyte to be detected to at least three binders with a certain affinity. Concerning the interaction between binder molecules and target molecules or molecules of interest, the affinity constant is preferably greater than 108M’1. In one embodiment of the invention such an assay is a sandwich immunoassay using any kind of detection technology including but not restricted to chemiluminescent label, electrochemiluminescence label, enzyme label, enzyme-amplified chemiluminescence label, fluorescence label, radioactive label and immunogold preferably a fully automated assay.

[0110] In one embodiment of the invention such an assay is an enzyme labeled sandwich immunoassay. Examples of automated or fully automated assay comprise assays that may be used for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Advia Centauer®, Siemens Immulite ®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®, Boditech AFIAS®, Ortho Vidas®, Diasorin LIASION®, Beckman Dxl®, Lumira Dx®, MeMed Key®, Werfen BioFlash®, BioRad BioPlex®.

[0111] A variety of immunoassay formats are known and may be used for the assays and methods of the present invention, these include radioimmunoassay’s ("RIA"), homogeneous enzyme-multiplied immunoassays ("EMIT"), enzyme-linked immunoadsorbent assays ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), chemiluminescence- (“CLIA”), electrochemiluminescence- (“ECLIA”) and fluorescence-immunoassays, Luminex-based bead arrays, protein microarray assays, and rapid test formats such as for instance immunochromatographic strip tests.

[0112] One embodiment of the present invention is a point-of-care (POC) device for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) comprising at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119- 159 (SEQ ID No. 6) in a sample of bodily fluid.

[0113] A POC-test is a test technology which allows performing the test within less than 1 hour near the patient without the requirement of a fully automated assay system. One example for this technology is the immunochromatographic test technology.

[0114] In one embodiment the assay for the determination of the level of proEnk fragment 119-159 may be performed as a test strip assay. In an exemplary test strip device, a test sample application pad is optionally attached to one end of a porous strip. The strip contains an immobilized antibody which will bind to and thereby immobilize proEnk fragment 119-159 at a predetermined site for subsequent detection. Optionally, the device may include an end of assay indicator which is positioned at the distal end of the test strip away from the test sample contact site. The end of assay indicator produces a detectable signal upon contact with the test sample or an assay reagent thereby indicating that the assay is complete.

[0115] A test sample application pad may be a portion of the porous strip itself or a material in fluidflow contact with the end of the porous strip, referred to as the proximal end, such that the test sample can pass or migrate from the application pad to the porous strip.

[0116] Fluid-flow contact can include physical contact of the application pad to the porous strip as well as the separation of the application pad from the porous strip by an intervening space or additional material which still allows fluid to flow between the application pad and the porous strip. Substantially all of the application pad can overlap the porous strip to enable the test sample to pass through substantially any part of the application pad to the proximal end of the porous strip. Alternatively, only a portion of the application pad might be in fluid-flow contact with the porous strip. The application pad can be any material which can transfer the test sample to the porous strip.

[0117] The porous strip of the assay device can be any suitably absorbent, porous, bibulous, chromatographic or capillary possessing material through which a test sample containing the analyte can be transported by a capillary or wicking action. Natural, synthetic, or naturally occurring materials that are synthetically modified, can be used as the porous strip including, but not limited to: cellulose materials such as paper, cellulose, and cellulose derivatives such as cellulose acetate and nitrocellulose; fiberglass; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon); porous gels such as silica gel, agarose, dextran, and gelatin; porous fibrous matrixes; starch based materials, such as crosslinked dextran chains; ceramic materials; films of polyvinyl chloride and combinations of polyvinyl chloride-silica; and the like. The porous strip should not interfere with the production of a detectable signal. The porous strip should have a reasonable inherent strength, or strength can be provided by means of a supplemental support.

[0118] In one embodiment of the invention at least one of said three binders is labeled in order to be detected.

[0119] The preferred detection methods comprise immunoassays in various formats such as for instance radioimmunoassay’s ("RIA"), homogeneous enzyme-multiplied immunoassays ("EMIT"), enzyme-linked immunoadsorbent assays ("ELISA"), enzyme-amplified chemiluminescence assays, apoenzyme reactivation immunoassay ("ARIS"), chemiluminescence- (“CLIA”), electrochemiluminescence- (“ECLIA”) and fluorescence-immunoassays, Luminex-based bead arrays, protein microarray assays, and rapid test formats such as for instance immunochromatographic strip tests.

[0120] In a preferred embodiment said label is selected from the group comprising chemiluminescent label, electrochemiluminescence label, enzyme-amplified chemiluminescence label, enzyme label, fluorescence label, radioactive label, immunogold.

[0121] In another embodiment, said labeling system comprises rare earth cryptates or rare earth chelates in combination with fluorescence dye or chemiluminescence dye, particularly a dye of the cyanine type.

[0122] In the context of the present invention, fluorescence based assays comprise the use of dyes, which may for instance be selected from the group comprising FAM (5-or 6-carboxyfluorescein), VIC, NED, Fluorescein, Fluorescein-isothiocyanate (FITC), IRD-700 / 800, Cyanine dyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, Xanthen, 6-Carboxy-2’,4’,7’,4,7-hexachlorofluorescein (HEX), TET, 6-Carboxy-4’,5’-dichloro-2’,7’-dimethodyfluorescein (JOE), N,N,N’,N’-Tetramethyl-6-carboxy- rhodamine (TAMRA), 6-Carboxy-X-rhodamine (ROX), 5-Carboxyrhodamine-6G (R6G5), 6- carboxyrhodamine-6G (RG6), Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes, such as BODIPY TMR, Oregon Green, Coumarines such as Umbelliferone, Benzimides, such as Hoechst 33258; Phenanthridines, such as Texas Red, Yakima Yellow, Alexa Fluor, PET, Ethidiumbromide, Acridinium dyes, Carbazol dyes, Phenoxazine dyes, Porphyrine dyes, Polymethin dyes, and the like. The fluorescent labels can be conjugated to an aldehyde group comprised in target molecule.

[0123] Luminescent dyes or labels can be further subcategorized into chemiluminescent and electrochemiluminescent dyes. The different classes of chemiluminogenic labels include luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives. For immunodiagnostic procedures predominantly acridinium based labels are used

[0124] In the context of the present invention, chemiluminescence based assays comprise the use of dyes, based on the physical principles described for chemiluminescent materials in (Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, J. I. Kroschwitz; editor, M. Howe- Grant, John Wiley & Sons, 1993, vol.15, p. 518-562, incorporated herein by reference, including citations on pages 551-562). Chemiluminescent label may be luminol label, acridinium ester label, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives, steroid labels involving isoluminol labels and the like. Preferred chemiluminescent dyes are acridiniumesters.

[0125] Enzyme labels may be lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), acidic phosphatase, glucose-6-phosphate dehydrogenase, horse radish peroxidase (HRP) and so on.

[0126] Alkaline phosphatase (ALP) is an enzyme used in combination with a colorimetric or chemiluminescent reagent in immunoassays. Typically, upon catalysis of a substrate by ALP, chemiluminescent or colored fluorescent signals that can be measured and quantified are produced.

[0127] ALP is a homodimer. In the active site of each monomer, there are three distinct metal binding sites (Ml, M2, and M3 sites). The catalytic activity of ALP is modulated by the metal content within these metal binding sites. Peak enzymatic activity is achieved when two of these sites (Ml and M2) are occupied by Zn2+and the third site (M3) is occupied by Mg2+.

[0128] Substrates that react with ALP include, for example, a combination of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chl oro-3 -indolyl phosphate (BCIP), p-Nitrophenyl Phosphate (PNPP), and Lumigen PPD (as found in LUMLPHOS 530 and LUMI-PHOS Plus (Lumigen, Inc., Southfield, MI)).

[0129] In some embodiments, the chemiluminescent substrate includes a chemiluminescent substrate and formulations thereof. In some aspects, the chemiluminescent substrate may include LUMIGEN APS-5 (Lumigen, Inc., Southfield, MI).

[0130] Electrochemiluminescense (ECL) proved to be very useful in analytical applications as a highly sensitive and selective method. It combines analytical advantages of chemiluminescent analysis (absence of background optical signal) with ease of reaction control by applying electrode potential. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescense is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. In ECL assays the labels of major relevance used as electrochemiluminescent labels are the Ruthenium- and the Iridium-based electrochemiluminescent complexes, respectively. Typical ECL immunoassays exploit tris(2,2'- bipyridine) ruthenium(II) ([Ru(bpy)3]2+) and tri-n-propylamine (TPrA) as luminophore and coreactant, respectively. The latter is a sacrificial molecular species that upon oxidation, undergoes an irreversible chemical step to generate strongly reducing radicals.

[0131] For the purposes of the present description, the term "solid phase" may be used to include any vessel in which or on which the assay may be performed and includes, but is not limited to porous materials, nonporous materials, test tubes, wells, slides, resins or particles. More specifically said solid phase material may be selected from the group comprising magnetic particles, paramagnetic particles, plastics (e.g. polystyrene, polypropylene, polycarbonate), cyclic olefin copolymers, glass, cellulose, nitrocellulose polyacrylamide, dextran, agarose, metal, or silicone.

[0132] In some aspects, the solid phase comprises particles selected from the group comprising microparticles, coated particles, coated microparticles, paramagnetic particles, or paramagnetic microparticles. In some embodiments, the particles are coated with antibodies, monoclonal antibodies, or target-specific antibodies. When the particles include paramagnetic particles, the particles may be separated from other components of a composition by being subjected to a magnetic field.

[0133] In some embodiments, the microparticles comprises a paramagnetic or superparamagnetic material such as, for example, ferromagnetic iron oxide FerCh or Fe2Ch. The terms “paramagnetic” and “superparamagnetic” refer to materials that experience a force in a magnetic field gradient, but do not become permanently magnetized. In a specific embodiment, the support comprises iron in the form of maghemite, or Fe20s. In various embodiments, the mean diameter of the microparticle is in the range of 100 nm to 22,900 nm.

[0134] Exemplary ranges of particles include at least 0.1 pg and up to 100 pg, at least 0.1 pg and up to 150 pg, at least 5 pg and up to 100 pg, at least 10 pg and up to 100 pg, etc. In some cases, the reagent includes particles from about 0.1 pg to about 150 pg, from about 0.2 pg to about 140 pg, from about 0.3 pg to about 130 pg, from about 0.4 pg to about 120 pg , from about 0.5 pg to about 110 pg, from about 1.0 pg to about 100 pg, from about 1.5 pg to about 90 pg, from about 2.0 pg to about 80 pg, from about 2.5 pg to about 70 pg, from about 3 pg to about 60 pg, from about 4 pg to about 50 pg, from about 0. 1 pg to about 60 pg, from about 0.1 pg to about 50 pg, or from about 0.5 pg to about 50 pg.

[0135] Binders of protein or peptide origin (e.g. antibody, antibody fragments, non-Ig scaffold) are immobilized onto the solid phase by methods comprising: non-covalent physical adsorption (e.g. by electrostatic interaction or hydrophobic interaction), bioaffinity immobilization (e.g. avidin-biotin, streptavidin-biotin, protein A / G / L, His-tag and Ni2+-NTA, GST-tag and gluthatione, DNA hybridization, aptamers), covalent binding techniques (e.g. amine and N- hydroxysuccinimide) or a combination of said immobilization methods.

[0136] In one embodiment said at least one capture binder is immobilized to said solid phase by biotin- related interaction (e.g. biotin-avidin, biotin-streptavidin).

[0137] In one embodiment subject matter of the present invention is a method for the determination of the level of proEnk fragment 119-159 in a bodily fluid (SEQ ID No. 6), the method comprising: providing a sample of bodily fluid of a subject, determining the level of proEnk fragment 119-159 in said sample of bodily fluid, using at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0138] In another embodiment subject matter of the present invention is a method for the determination of the level of proEnk fragment 119-159 in a bodily fluid (SEQ ID No. 6), the method comprising: providing a sample of bodily fluid of a subject, determining the level of proEnk fragment 119-159 in said sample of bodily fluid, wherein determining the level of proEnk fragment 119-159 comprises steps of contacting said sample with at least three binders binding to three different nonoverlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0139] In yet another embodiment subject matter of the present invention is a method for the determination of the level of proEnk fragment 119-159 in a bodily fluid (SEQ ID No. 6), the method comprising: providing a sample of bodily fluid of a subject, determining the level of proEnk fragment 119-159 in said sample of bodily fluid, forming a reaction mixture in any order or concurrently by adding at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0140] In one embodiment the method comprises the steps of contacting said sample with at least two capture binders directed to two different non-overlapping regions of proEnk fragment 119-159 and at least one binder directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 which is labelled for detection.

[0141] In another embodiment the method comprises the steps of contacting said sample with at least two binders directed to two different non-overlapping regions of proEnk fragment 119-159 which are labelled for detection and at least one capture binder directed to a third region not overlapping with the first and second region of proEnk fragment 119-159.

[0142] In yet another embodiment the method comprises the steps of contacting said sample with at least one capture binder directed to one region of proEnk fragment 119-159, at least one binder directed to a second region of proEnk fragment 119-159 which is labelled for detection and at least one binder directed to a third region of proEnk 119-159 which is not labelled for detection and is not a capture binder, wherein said first, second and third region of proEnk fragment 119-159 are nonoverlapping regions.

[0143] The term “contacting said sample with ...” also means “contacting proEnk fragment 119-159 contained in said sample with ...”.

[0144] Said bodily fluid may be selected from the group comprising blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva. In one embodiment of the invention the bodily fluid is selected from the group comprising whole blood, blood plasma (e.g. EDTA-plasma, heparin- plasma), and blood serum.

[0145] In one embodiment of the methods for determining the level of proEnk fragment 119-159 in a sample of bodily fluid according to the present invention such assay is a sandwich assay, preferably a fully automated assay. It may be an ELISA fully automated or manual. It may be a so-called POC-test (point-of-care). Examples of automated or fully automated assay comprise assays that may be used for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Advia Centauer®, Siemens Immulite ®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®, Boditech AFIAS®, Ortho Vidas®, Diasorin LIASION®, Beckman Dxl®, Lumira Dx®, MeMed Key®, Werfen BioFlash®, BioRad BioPlex®. Examples of test formats are provided above.

[0146] In one embodiment of the methods for determining the level of proEnk fragment 119-159 in a sample of bodily fluid according to the present invention at least one of said three binders is labeled in order to be detected. Examples of labels are provided above. In one embodiment of the methods for determining the level of proEnk fragment 119-159 in a sample of bodily fluid according to the present invention at least one of said three binders is bound to a solid phase. Examples of solid phases and solid phase materials are provided above.

[0147] In one embodiment of the methods for determining the level of proEnk fragment 119-159 in a sample of bodily fluid according to the present invention said label is selected from the group comprising chemiluminescent label, electrochemiluminescence label, enzyme-amplified chemiluminescence label, enzyme label, fluorescence label, radioactive label, immunogold.

[0148] In one embodiment of the methods for determining the level of proEnk fragment 119-159 in a sample of bodily fluid according to the present invention said solid phase is selected from the group comprising porous materials, nonporous materials, test tubes, wells, slides, resins or particles.

[0149] In one embodiment of the methods for determining the level of proEnk fragment 119-159 in a sample of bodily fluid according to the present invention said solid phase material may be selected from the group comprising magnetic particles, paramagnetic particles, plastics (e.g. polystyrene, polypropylene, polycarbonate), cyclic olefin copolymers, glass, cellulose, nitrocellulose polyacrylamide, dextran, agarose, metal, or silicone.

[0150] In one embodiment the assay comprises three binders, preferably antibodies which are all present as dispersions in a liquid reaction mixture, wherein: a first labelling component is attached to the first binder molecule, wherein said first labelling component is part of a labelling system based on fluorescence- or chemiluminescence-quenching or amplification, and a second labelling component of said marking system is attached to a second binder molecule, and a third binder molecule is unlabeled, wherein upon binding of both labelled binder molecules to the analyte in the presence of said unlabeled binder molecule, a measurable signal is generated that allows for the detection of the formed sandwich complexes in the solution comprising the sample, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0151] In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first binder molecule is bound to a solid phase, a second binder molecule is bound a detectable label, and a third binder molecule is unlabeled, wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed sandwich complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0152] In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first and a second binder molecule is bound to a solid phase, a third binder molecule is bound a detectable label, and wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed sandwich complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0153] In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first binder molecule is bound to a solid phase, a second and third binder molecule is bound a detectable label, and wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed sandwich complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0154] In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first binder molecule is bound to a solid phase, wherein the solid phase material is magnetic or paramagnetic, a second binder molecule is bound to a detectable label, wherein said label is a chemiluminescent label or an enzyme-amplified chemiluminescent label and a third binder molecule is unlabeled, wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6). In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first and a second binder molecule is bound to a solid phase, wherein the solid phase material is magnetic or paramagnetic, a third binder molecule is bound to a detectable label, wherein said label is a chemiluminescent label or an enzyme-amplified chemiluminescent label, wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0155] In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first binder molecule is bound to a solid phase, wherein the solid phase material is magnetic or paramagnetic, a second and third binder molecule is bound to a detectable label, wherein said label is a chemiluminescent label or an enzyme-amplified chemiluminescent label, wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0156] In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first binder molecule is bound to a solid phase, wherein the solid phase material is magnetic or paramagnetic, a second binder molecule is bound to a detectable label, wherein said label is an electrochemiluminescent label and a third binder molecule is unlabeled, wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0157] In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first and a second binder molecule is bound to a solid phase, wherein the solid phase material is magnetic or paramagnetic, a third binder molecule is bound to a detectable label, wherein said label is an electrochemiluminescent label, wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0158] In one embodiment the assay comprises three binders, preferably antibodies, wherein: a first binder molecule is bound to a solid phase, wherein the solid phase material is magnetic or paramagnetic, a second and third binder molecule is bound to a detectable label, wherein said label is an electrochemiluminescent label, wherein upon binding of all three binders to the analyte, a measurable signal is generated that allows for the detection of the formed complexes, and wherein the three binder molecules are directed to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

[0159] In one embodiment subject matter of the present invention is a kit for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) comprising at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6) in a sample of bodily fluid.

[0160] In yet another aspect, the disclosure relates to a kit for detecting proEnk 119-159 fragment in a sample. In embodiments of this aspect, a kit can comprise any of the binders, lables, substrates, enzymes, formulations or compositions comprising the binders, lables, substrates, enzymes, and formulations as generally described herein, and instructions or a label directing appropriate use. Optionally, a kit may also include one or more containers, reagents, reactants, and / or assay diluents, or other devices to facilitate use. The disclosure contemplates that all or any subset of the components for conducting research assays and / or diagnostic assays may be included in the kit. Similarly, the kit may include instructions for making one or more assay solutions comprising one or more of the assay biological samples, binders, lables, substrates, enzymes, formulations, or compositions thereof, under suitable conditions. In some additional example embodiments, a kit may comprise a solution, or a dried or lyophilized preparation of one or more kit components, and instructions for preparing the solution, or dried or lyophilized preparation for use (e.g., for reconstituting a lyophilized or dried product, dispensing and / or diluting a solution, etc.). The disclosure also encompasses a finished packaged and labeled product (e.g., as a kit, or as one or more parts of a kit). Such an article of manufacture includes the appropriate unit form (e.g., concentrated assay formulations, ready to use assay formulations, etc.) in an appropriate vessel or container such as a glass vial or other container that is typically sterile and sealed. In some example embodiments containers can include, but are not limited to, vials, bottles, and / or pre-filled syringes, and the like. Optionally associated with such kit(s) and / or containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of the kit and / or assay components, which notice can reflect approval by an agency of the manufacture, use or sale for human use / diagnosis

[0161] The kit comprising one or more of a binder, a label, substrate, enzyme; and / or an assay reagent, or one or more compositions comprising the same, and in accordance with the aspects and embodiments of the disclosure. In some embodiments, the kits can further comprise additional assay reagents such as assay buffers, assay diluents, pH adjusting agents, or additional reporter molecules (e.g., for the indirect or detection of the target analyte or antigen). The kits can also comprise instructions for use in the practice of any one of the assays or methods disclosed herein.

[0162] The methods, compositions, and kits disclosed herein may be used in combination with integrated system platforms. For example, the methods, compositions, and kits may be used in combination systems commercially marketed, e.g., by Beckman Coulter such as, for example, the immunoassay analyzers including. Access 2+, UniCel Dxl 600, UniCel, Dxl 800, or Dxl 9000 immunoassay analyzers. In some exemplary embodiments, the integrated system platform is the Dxl 9000 immunoassay analyzer. The methods, compositions, and kits disclosed herein can also be used with one or more sample preparation system platforms.

[0163] Similarly, the methods, compositions, and kits disclosed herein may be used with point-of-care system platforms as well as other available assay platforms. As such, the methods, compositions, and kits of the disclosure can be readily adapted for use with any number of devices, assay platforms, and instrumentation such as, for example, handheld fluorescence detectors, microfluidic devices, enzymatic detection systems, immunochromatographic strips, and lateral flow devices. With the above context, the following consecutively numbered embodiments provide further specific aspects of the invention:

[0164] 1. Use of at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (SEQ ID No. 6) for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a sample of bodily fluid.

[0165] 2. The use according to embodiment 1, wherein the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) is a specific determination.

[0166] 3. The use according to embodiments 1 or 2, wherein said three different non-overlapping epitopes of proEnk fragment 119-159 are within the N-terminal region that is amino acid 119 to 133 (SEQ ID No. 2), within the mid-regional region that is amino acid 129 to 155 (SEQ ID No. 7) and within the C-terminal region that is amino acid 152 to 159 (SEQ ID No. 5), wherein each of said epitopes comprises at least 4 or 5 amino acids.

[0167] 4. The use according to embodiments 1 to 3, wherein said binder binding to the N-terminal region recognizes and binds to the N-terminal end (binds to amino acid 119, which is aspartic acid) of proEnk fragment 119-159 and wherein said binder binding to the C- terminal region recognizes and binds to the C-terminal end (binds to amino acid 159, which is serine) of proEnk fragment 119-159.

[0168] 5. The use according to embodiments 1 to 4, wherein said binder is an antibody, an antibody fragment, an aptamer or a non-Ig-scaffold.

[0169] 6. The use according to embodiments 1 to 5, wherein said at least three binders are utilized in an immunoassay.

[0170] 7. The use according to embodiment 6, wherein said immunoassay is a sandwich immunoassay.

[0171] 8. The use according to embodiments 1 to 7, wherein at least two of said binders directed to two different non-overlapping regions of proEnk fragment 119-159 are used as capture binders and at least one of said binders is directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 is labelled for detection. The use according to embodiments 1 to 7, wherein at least two of said binders directed to two different non-overlapping regions of proEnk fragment 119-159 are labelled for detection and at least one of said binders directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 is a capture binder. The use according to embodiments 1 to 7, wherein at least one of said binders directed to one region of proEnk fragment 119-159 is a capture binder, at least one of said binders directed to a second region of proEnk fragment 119-159 is labelled for detection and at least one of said binders directed to a third region of proEnk 119-159 is not labelled for detection and is not a capture binder, wherein said first, second and third region of proEnk fragment 119-159 do not overlap. The use according to embodiments 1 to 10, wherein said at least one capture binder is immobilized to a solid phase. The use according to embodiment 11, wherein said solid phase is selected from the group of material comprising magnetic particles, paramagnetic particles, plastics (e.g. polystyrene, polypropylene, polycarbonate), cyclic olefin copolymers, glass, cellulose, nitrocellulose polyacrylamide, dextran, agarose, metal, or silicone. The use according to embodiment 11, wherein said at least one capture binder is immobilized to said solid phase by non-covalent physical adsorption, bioaffinity immobilization techniques or covalent binding techniques. The use according to embodiment 13, wherein said at least one capture binder is immobilized to said solid phase by biotin-related interaction (e.g. biotin-avidin, biotinstreptavidin). The use according to embodiments 1 to 10, wherein said label is selected from the group comprising luminescent label, chemiluminescent label, electrochemiluminescence label, enzyme label, enzyme-amplified chemiluminescence label, fluorescence label, radioactive label, immunogold. The use according to embodiment 15, wherein said labeling system comprises rare earth cryptates or rare earth chelates in combination with fluorescence dye or chemiluminescence dye, in particular a dye of the cyanine type. The use according to embodiment 15, wherein said chemiluminescent label is selected from the group comprising luminol label, acridinium ester label, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives, steroid labels involving isoluminol labels. The use according to embodiment 15, wherein said enzyme label is selected from the group comprising lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), acidic phosphatase, glucose-6-phosphate dehydrogenase, horse radish peroxidase (HRP). The use according to embodiments 1 to 11, wherein said binder exhibits an affinity constant to proEnk fragment 119-159 of at least 107M’1, preferred 108M’1, preferred affinity constant is greater than 109M’1, most preferred greater than IO10M'1. The use according to embodiment 1, wherein said bodily fluid is selected from the group comprising blood, cerebrospinal fluid, urine and saliva. The use according to embodiment 20, wherein the blood sample is selected from the group comprising whole blood, blood plasma (e.g. EDTA-plasma, heparin-plasma) and blood serum. A kit for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) comprising at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6) in a sample of bodily fluid. A kit according to embodiment 22, wherein the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) is a specific determination. A kit according to embodiment 22 and 23, wherein said three different non-overlapping epitopes of proEnk fragment 119-159 are within the N-terminal region that is amino acid 119 to 133 (SEQ ID No. 2), within the mid-regional region that is amino acid 129 to 155 (SEQ ID No. 7) and within the C-terminal region that is amino acid 152 to 159 (SEQ ID No. 5), wherein each of said epitopes comprises at least 4 or 5 amino acids. A kit according to embodiment 24, wherein said binder binding to the N-terminal region recognizes and binds to the N-terminal end (binds to amino acid 119, which is aspartic acid) of proEnk fragment 119-159 and wherein said binder binding to the C-terminal region recognizes and binds to the C-terminal end (binds to amino acid 159, which is serine) of proEnk fragment 119-159. A kit according to embodiments 22 to 25, wherein said binder is an antibody, an antibody fragment, an aptamer or a non-Ig-scaffold. A kit according to embodiments 22 to 26, wherein said at least three binders are utilized in an immunoassay. A kit according to embodiment 27, wherein said immunoassay is a sandwich immunoassay. A kit according to embodiments 22 to 28, wherein at least two of said binders directed to two different regions of proEnk fragment 119-159 are capture binders and at least one of said binders is directed a third region of proEnk fragment 119-159 is labelled for detection. A kit according to embodiments 22 to 28, wherein at least two of said binders directed to two different non-overlapping regions of proEnk fragment 119-159 are labelled for detection and at least one of said binders is directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 is a capture binder. A kit according to embodiments 22 to 28, wherein at least one of said binders directed to one region of proEnk fragment 119-159 is a capture binder, at least one of said binders directed to a second region of proEnk fragment 119-159 is labelled for detection and at least one of said binders directed to a third region of proEnk fragment 119-159 is not labelled for detection and is not a capture binder, wherein the first, second and third region of proEnk 119-159 do not overlap. A kit according to embodiments 22 to 31, wherein said capture binder is immobilized to a solid phase. A kit according to embodiment 32, wherein said solid phase is selected from the group of material comprising magnetic particles, paramagnetic particles, plastics (e.g. polystyrene, polypropylene, polycarbonate), cyclic olefin copolymers, glass, cellulose, nitrocellulose polyacrylamide, dextran, agarose, metal, or silicone. A kit according to embodiment 33, wherein said capture binder is immobilized to said solid phase by non-covalent physical adsorption, bioaffinity immobilization techniques or covalent binding techniques. A kit according to embodiment 34, wherein said at least one capture binder is immobilized to said solid phase by biotin-related interaction (e.g. biotin-avidin, biotin-streptavidin). A kit according to embodiments 22 to 31, wherein said label is selected from the group comprising luminescent label, chemiluminescent label, electrochemiluminescence label, enzyme label, enzyme-amplified chemiluminescence label, fluorescence label, radioactive label, immunogold. A kit according to embodiment 36, wherein said labeling system comprises rare earth cryptates or rare earth chelates in combination with fluorescence dye or chemiluminescence dye, in particular a dye of the cyanine type. A kit according to embodiment 36, wherein said chemiluminescent label is selected from the group comprising luminol label, acridinium ester label, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives, steroid labels involving isoluminol labels. A kit according to embodiment 36, wherein said enzyme label is selected from the group comprising lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), acidic phosphatase, glucose-6-phosphate dehydrogenase, horse radish peroxidase (HRP). A kit according to embodiments 22 to 32, wherein said binder exhibits an affinity constant to proEnk fragment 119-159 of at least 107M’1, preferred 108M’1, preferred affinity constant is greater than 109M’1, most preferred greater than IO10M'1. The use according to embodiment 22, wherein said bodily fluid is selected from the group comprising blood, cerebrospinal fluid, urine and saliva. The use according to embodiment 41, wherein the blood sample is selected from the group comprising whole blood, blood plasma (e.g. EDTA-plasma, heparin-plasma) and blood serum. A method for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid, the method comprising: providing a sample of bodily fluid of a subject, determining the level of proEnk fragment 119-159 in said sample of bodily fluid, using at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6). A method according to embodiment 43, wherein the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) is a specific determination. A method according to embodiment 43, wherein said three different non-overlapping epitopes of proEnk fragment 119-159 are within the N-terminal region that is amino acid 119 to 133 (SEQ ID No. 2), within the mid-regional region that is amino acid 129 to 155 (SEQ ID No. 7) and within the C-terminal region that is amino acid 152 to 159 (SEQ ID No. 5), wherein each of said epitopes comprises at least 4 or 5 amino acids. A method according to embodiments 43 to 45, wherein said binder binding to the N- terminal region recognizes and binds to the N-terminal end (binds to amino acid 119, which is aspartic acid) of proEnk fragment 119-159 and wherein said binder binding to the C- terminal region recognizes and binds to the C-terminal end (binds to amino acid 159, which is serine) of proEnk fragment 119-159. A method according to embodiment 43, wherein said sample is selected from the group comprising blood, cerebrospinal fluid, urine and saliva. A method according to embodiment 47, wherein the blood sample is selected from the group comprising whole blood, blood plasma (e.g. EDTA-plasma, heparin-plasma) and blood serum. A method according to embodiment 43, wherein determining the level of proEnk fragment 119-159 comprises the steps of contacting said sample with at least two capture binders directed to two different non-overlapping regions of proEnk fragment 119-159 and at least one binder directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 which is labelled for detection.

[0172] 50. A method according to embodiment 43, wherein determining the level of proEnk fragment 119-159 comprises the steps of contacting said sample with at least two binders directed to two different non-overlapping regions of proEnk fragment 119-159 which are labelled for detection and at least one capture binder directed to a third region not overlapping with the first and second region of proEnk fragment 119-159.

[0173] 51. A method according to embodiment 43, wherein determining the level of proEnk fragment 119-159 comprises the steps of contacting said sample with at least said one capture binder directed to one region of proEnk fragment 119-159, at least said one binder directed to a second region of proEnk fragment 119-159 which is labelled for detection and at least said one binder directed to a third region of proEnk 119-159 which is not labelled for detection and is not a capture binder, wherein the first, second and third region of proEnk 119-159 do not overlap.

[0174] 52. A method according to embodiments 49 to 51, wherein said capture binder is immobilized to a solid phase.

[0175] 53. A method according to embodiment 52, wherein said solid phase is selected from the group of material comprising magnetic particles, paramagnetic particles, plastics (e.g. polystyrene, polypropylene, polycarbonate), cyclic olefin copolymers, glass, cellulose, nitrocellulose polyacrylamide, dextran, agarose, metal, or silicone.

[0176] 54. A method according to embodiments 52 and 53, wherein said capture binder is immobilized to said solid phase by non-covalent physical adsorption, bioaffinity immobilization techniques or covalent binding techniques.

[0177] 55. A method according to embodiment 54, wherein said capture binder is immobilized to said solid phase by biotin-interaction (e.g. biotin-avidin, biotin-streptavidin).

[0178] 56. A method according to embodiments 49 to 51, wherein said label is selected from the group comprising luminescent label, electrochemiluminescence label, chemiluminescent label, enzyme-amplified chemiluminescence label, enzyme label, fluorescence label, radioactive label, immunogold. A method according to embodiment 56, wherein said labeling system comprises rare earth cryptates or rare earth chelates in combination with fluorescence dye or chemiluminescence dye, in particular a dye of the cyanine type. A method according to embodiment 56, wherein said chemiluminescent label is selected from the group comprising luminol label, acridinium ester label, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives, steroid labels involving isoluminol labels. A method according to embodiment 56, wherein said enzyme label is selected from the group comprising lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), acidic phosphatase, glucose-6-phosphate dehydrogenase, horse radish peroxidase (HRP). A method according to embodiments 53 to 44 and embodiments 49 to 55, wherein said binder exhibits an affinity constant to proEnk fragment 119-159 of at least 107M’1, preferred 108M’1, preferred affinity constant is greater than 109M’1, most preferred greater than IO10M’1.

[0179] EXAMPLES

[0180] Example 1 - Generation and characterization of antibodies

[0181] Peptides / conjugates for immunization:

[0182] Peptides for immunization were synthesized (JPT Technologies, Berlin, Germany) with an additional cystein residue for conjugation of the peptides to bovine serum albumin (BSA) (table 2). The peptides were covalently linked to BSA by using Sulfo-SMCC (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio.

[0183] The antibodies were generated according to the following method:

[0184] A BALB / c mouse was immunized with 100 pg peptide-BSA-conjugate at day 0 and 14 (emulsified in 100 pl complete Freund’s adjuvant) and 50 pg at day 21 and 28 (in 100 pl incomplete Freund’s adjuvant). Three days before the fusion experiment was performed, the animal received 50 pg of the conjugate dissolved in 100 pl saline, given as one intraperitoneal and one intravenous injection. Splenocytes from the immunized mouse and cells of the myeloma cell line SP2 / 0 were fused with 1 ml 50 % polyethylene glycol for 30 s at 37 °C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium (RPMI 1640 culture medium supplemented with 20 % fetal calf serum and HAT-supplement). After two weeks the HAT medium is replaced with HT Medium for three passages followed by returning to the normal cell culture medium. The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion. The positive tested microcultures were transferred into 24- well plates for propagation. After retesting the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined (Lane et al. 1985. J. Immunol. Meth. 81: 223-228; Ziegler et al 1996. Horm. Metab. Res. 28: 11-15).

[0185] Monoclonal antibody production

[0186] Antibodies were produced via standard antibody production methods (Marx et al.. Monoclonal Antibody Production (1997), ATLA 25, 121) and purified via Protein A-chromatography. The antibody purities were > 95 % based on SDS gel electrophoresis analysis. Table 2: Immunization peptides and antibodies against proEnk 119-159

[0187] (NT = N-terminal; N-MR = N-terminal side of mid-regional proEnk; C-MR = C-terminal side of mid-regional proEnk; MR = mid-regional; CT = C-terminal)

[0188] Labeling of antibodies All antibodies were labelled with acridinium ester according to the following procedure:

[0189] Purified anti -proEnk 119-159 antibodies as listed in table 2 (1 g / L) were labeled by incubation in 10% labeling buffer (500 mM sodium phosphate, pH 8.0) with 1 :5 molar ratio of MACN- Acridinium-NHS-ester (1 g / L, InVent GmbH) for 20 min at 22°C. After adding 5% 1 M Tris / HCl, pH 8.0, for 10 minutes labeled antibody was separated from free label via CentriPure P10 columns (emp Biotech GmbH) and by Gel -filtration HPLC on Protein KW-803 (Shodex, Showa DEnko

[0190] Europe GmbH, Munich, Germany). The purified labelled antibody was diluted in (300 mmol / 1 potassium phosphate, 100 mmol / 1 NaCl, 10 mmol / 1 Na-EDTA, 5 g / 1 bovine serum albumin, pH 7.0). The final concentration was approx. 10 ng of labeled antibody per 200 pL. Acridiniumester chemiluminescence was measured by using Centro 960 microtiter plate luminescence reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).

[0191] Solid phase antibody (coating of antibody):

[0192] White polystyrene microtiter plates (Greiner Bio-One International AG, Austria) were coated (18 h at 4°C) with the respective antibodies (1 pg / 0.2 mL per well, 200 mmol / L Tris / HCl, 100 mmol / L NaCl, pH 7.8). After blocking with 30 g / L Karion, 5 g / L BSA (protease free), 6.5 mmol / L monopotassium phosphate, 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum dried.

[0193] Antibody specificity

[0194] Antibody cross-reactivities were determined as follows: Ipg peptide in 200 pl PBS, pH 7.4 was pipetted into polystyrene microtiter plates and incubated for Ih at room temperature. After incubation the plates were washed 5 times (each 300pl) using 5% BSA in PBS, pH 7.4. Each of the labelled antibodies were added (200 pl in PBS, pH 7.4, 800.000 RLU / 200 pl) and incubated for 2h at room temperature. After washing 5 times (each 300pl of washing solution (20 mmol / 1 PBS, pH 7.4, 0.1 % Triton X 100), the remaining luminescence (labelled antibody) was quantified using the Centro 960 microtiter plate luminescence reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). ProEnk 119-159-peptide was used as reference substance (100%). Results are shown as mean + / - SD for each group of antibody binding region. The cross-reactivities of the different antibodies are listed in table 3.

[0195] Table 3: cross-reactivities of proEnk 119-159-antibodies

[0196] All antibodies bound the proEnk 119-159 peptide, comparable to the peptides which were used for immunization. proEnk 119-159 immunoassay:

[0197] 50 pl of sample (or calibrator) was pipetted into polystyrene microtiter plates, after adding labelled antibody (200 pl), the plates were incubated for 2 h at 18-25 °C. Unbound tracer was removed by washing 5 times (each 350 pl) with washing solution (20 mmol / 1 PBS, pH 7.4, 0.1 % Triton X-100). Well-bound chemiluminescence was measured for Is per well by using the Centro LB 960 microtiter plate luminescence reader (Berthold Technologies GmbH & Co. KG, Germany).

[0198] Using a fixed concentration of 1000 pmol / L of proEnk 119-159, the signal (RLU at 1000 pmol proEnk 119-159 / 1) to noise (RLU without proEnk 119-159) ratio of different antibody combinations was calculated. All antibodies were able to generate a sandwich complex with any other antibody, except from a combination detecting specifically the N-terminal and the C-terminal end of proEnk 119-159, respectively. Signal-to-noise-ratios of specific antibody combinations with highest values are given in table 4 (the other antibodies as listed in table 2, were tested as well and gave comparable, but slightly lower results). The strongest signal-to-noise-ratio (best sensitivity) was generated by combining the N-MR-proEnk 119-159 antibodies and CT-proEnk 119-159 antibodies.

[0199] Table 4: Signal-to-noise-ratio of antibody combinations

[0200] Calibration:

[0201] The assay was calibrated, using dilutions of synthetic proEnk 119-159 (JPT Peptide Technologies GmbH). The lowest calibrator did not contain any proEnk 119-159 peptide. The calibrators were lyophilized in 20 mmol K2PO4, 6 mM Na-EDTA, 5 g / L BSA, 100 pM Leupeptin, 50 pM Amastatin, pH 8.0, and reconstituted in heat-inactivated horse serum (Gibco® Thermofisher Scientific, Boston, USA) prior to use. Epitope mapping of antibodies a) C-terminal anti-proEnk 119-159 antibodies

[0202] The epitope specificity of anti-proEnk mAb’s binding to the C-terminus (amino acids 152-159) of proEnk fragment 119-159 (as listed in table 2) was assessed as follows: Purified antibody was labeled with MACN-acridinium-NHS(N-hydroxysuccimimide)-ester as described above. Polystyrene microtiter plates (Greiner) were coated with streptavidin (Sigma Aldrich) as follows: 250 pL of a 10 pg streptavidin / mL solution in 50 mM Tris, 100 mM NaCl, pH 7.8, were pipetted into each well. After 20 h incubation at 22°C, solutions were aspirated, and 300 pL 10 mmol / L Na-phosphate, 3% Karion FP, 0.5% BSA, pH 6.5, were added for 1 h at 22 °C. After aspiration, biotinylated peptides, as listed in table 5, were immobilized on microtiter plates. Peptides were diluted to a final concentration of 1 mg / L in PBS buffer containing 5 g / L BSA, and 250 pL were pipetted into each well. After 3 h incubation at 22°C and orbital agitation at 600 rpm, solutions were aspirated, and 300 pL 10 mmol / L Na-phosphate, 3% Karion FP, 0.5% BSA, pH 6.5, were added for Ih at 22°C. The solution was aspirated. The respective chemiluminescence-labeled antibodies were diluted in buffer (300 mmol / L K-phosphate, 100 mmol / L NaCl, 10 mmol / L Na- EDTA, 5 g / L BSA, 1 g / L unspecific mouse and bovine IgG, 0.1% Na-azide, pH 7.4) to a concentration of 10 ng / 200 pL per well, and 200 pL of this tracer solutions were added and incubated for 3 h at 22 °C and orbital agitation at 600 rpm. Then the microtiter plates were washed 5 times with 300 pL washing solution each per well, and bound chemiluminescence was measured for 1 s per well with a Centro 960 microtiter plate luminescence reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).

[0203] Table 5: Epitope mapping of anti-proEnk mAb's binding to the C-terminus (amino acids 152-159) of proEnk fragment 119-159.

[0204] The different N-terminal biotinylated peptides were immobilized on microtiter plates coated with streptavidin. The binding (B) of labeled antibody on microtiter plates with the indicated peptides was analyzed and compared to the binding of peptide PENK 145-159, which represents the very C -terminal moiety of the native analyte pro-Enk 119-159. b) N-terminal anti-proEnk 119-159 antibodies

[0205] The epitope specificity of anti-proEnk mAb’s binding to the N-terminus (amino acids 119-131) of proEnk fragment 119-159 (as listed in table 2) was assessed as described above. Results of Epitope mapping are shown in table 6.

[0206] Table 6: Epitope mapping of anti-proEnk mAb’s binding to the N-terminus (amino acids 119-131) of proEnk 119-159.

[0207] The different C-terminal biotinylated peptides were immobilized on microtiter plates coated with streptavidin. The binding (B) of labeled antibody on microtiter plates with the indicated peptides was analyzed and compared to the binding of peptide PENK 119-130, which represents the very C-terminal moiety of the native analyte pro-Enk 119-159.

[0208] Example 2 - Immunoassay using three antibodies with different non-overlapping binding regions

[0209] 2.1. Addition of unbound and unlabeled antibodies (“cold”)

[0210] As shown in example 1 table 4, there was no signal when using two antibodies specifically directed to the N- and C-terminus, respectively. However, if using an antibody against the N-terminal portion or C-terminal portion of proEnk fragment 119-159 in combination with an antibody against the mid-regional portion of proEnk fragment 119-159, a signal was measurable.

[0211] 2.1.1. Sandwich assay using “cold” antibodies directed to the mid-regional part of proEnk fragment 119-159 Surprisingly, when an unbound and unlabeled antibody directed to the mid-regional part of proEnk fragment 119-159 (AK2334) was added to the reaction mixture, a signal could be generated with proEnk fragment 119-159 standard concentrations (50 to 2.500 pmol / L) and EDTA-plasma samples from human blood donors with both, antibodies against the N-terminal part of proEnk fragment 119-159 (AK2259) as solid phase and antibodies against the C-terminal part of proEnk fragment 119-159 (AK1879) as tracer antibody (see table 7) and vice-versa (table 8), respectively. Different blood sample matrices gave similar measuring values. Serum, EDTA-plasma and heparin-plasma was taken in parallel from five healthy blood donors. EDTA-plasma was used as reference and set 100%. Serum samples gave 107.8% + / - 5.7% and heparin-plasma samples 96.5% + / - 3.2 % when compared to EDTA-plasma samples.

[0212] Table 7: Measuring values of a sandwich assay using an antibody against the N-terminal part of proEnk fragment 119-159 (AK2259) as solid phase and an antibody against the C-terminal part of proEnk fragment 119-159 (AK1879) as tracer antibody with and without “cold” antibody against the mid-regional part of proEnk fragment 119-159 (AK2334) (n.d. not detectable)

[0213] Table 8: Measuring values (relative light units [RLU] minus RLU values of unspecific binding) of a sandwich assay using an antibody against the C-terminal part of proEnk fragment 119-159 (AK 1879) as solid phase and an antibody against the N-terminal part of proEnk fragment 119-159 (AK 2259) as tracer antibody with and without “cold” antibody against the mid-regional part of proEnk fragment 119-159 (AK 2334) (n.d. not detectable)

[0214] 2.1.2. Sandwich assay using a third “cold” antibody directed to the N-terminal part of proEnk fragment 119-159 In a next step, we tested, whether an unbound and unlabeled antibody is able to increase the binding of proEnk fragment 119-159 if a sandwich assay is used combining the N-MR-proEnk 119-159 antibodies (AK 2334) and CT-proEnk 119-159 antibodies (AK 1879) which generated the strongest signal-to-noise-ratio (best sensitivity) as described in example 1 table 4 (reference assay). Table 9 and Figure 1 show the RLU-values measured for calibrators containing defined concentrations of proEnk fragment 119-159. Using increasing concentrations of a “cold” third antibody in the reaction mixture resulted in significantly elevated measuring values and an increased sensitivity of the assay (as demonstrated by the increased calibrator ratios).

[0215] Table 9: Measuring values (relative light units [RLU]) of a sandwich assay using an antibody against the C-terminal part of proEnk fragment 119-159 (AK 1879) as solid phase and an antibody against the mid-regional part of proEnk fragment 119-159 (AK 2334) as tracer antibody without (Reference) and with “cold” antibody against the N-terminal part of proEnk fragment 119-159 (AK 2259) In a next attempt, human samples were measured using a sandwich assay with an antibody against the C-terminal part of proEnk fragment 119-159 (AK 1879) as solid phase and an antibody against the mid-regional part of proEnk fragment 119-159 (AK 2334) as tracer without (Reference) and with “cold” antibody against the N-terminal part of proEnk fragment 119-159 (AK 2259). Ipg “cold” antibody per well was used for all further experiments.

[0216] Table 10 shows the results of proEnk 119-159 calibrators without (Reference) and with the addition of “cold” antibody against the N-terminal part of proEnk fragment 119-159 (AK 2259). The addition of Ipg of AK2259 per well resulted in significantly higher RLU-values, especially in the lower concentration range.

[0217] Table 10: Measuring values for proEnk 119-159 calibrator

[0218] Table 11 shows measuring values and corresponding concentration values of EDTA-plasma and whole blood samples. The addition of “cold” antibody AK2259 resulted in elevated measuring values (RLU’s) for both, EDTA-plasma and whole blood, respectively. The corresponding concentration values calculated using respective calibrators (with and without “cold” antibody) resulted in concentration values as given in table 11, showing lower concentrations in whole blood as compared to EDTA-plasma.

[0219] Table 11 : Measuring values and corresponding concentration values of EDTA-plasma and whole blood samples

[0220] 2.2. Immunoassay using two antibodies as solid phase antibodies and a third antibody as tracer antibody

[0221] In a next step, an immunoassay was tested using two antibodies directed against two different regions of proEnk 119-159 as solid phase antibodies and a third antibody against a third region of proEnk 119-159 as tracer antibody.

[0222] 2.2.1. Microtiter plate sandwich immunoassay (direct coating of antibodies)

[0223] All coating and labelling procedures as well as the immunoassays were carried out as described in Example 1. For all immunoassays 2pg of antibodies were directly coated in total to each well (via non-covalent physical adsorption). A sandwich assay using only two antibodies, one antibody against the C-terminal part of proEnk fragment 119-159 (AK1879) as solid phase and a second antibody against the mid-regional part of proEnk fragment 119-159 (AK2334) as tracer was used as reference assay. Two other solid phase antibodies were tested (using the same tracer AK2334): one was also directed to the C-terminal part of proEnk fragment 119-159 (AK3278) and the other antibody was directed to the N-terminal part of proEnk fragment 119-159 (AK3271), respectively. Using AK3278 instead of AK1879 showed a slight increase in signals. If AK3271, which is directed against the N-terminal part of proEnk fragment 119-159, is used as solid phase, the signals were significantly increased compared to immunoassays using an antibody against the C-terminal portion of proEnk fragment 119-159. However, if using both, an antibody against the N-terminal part (AK3271) and against the C-terminal part (AK3278) of proEnk fragment 119-159, the signal increased up to ten-fold. This is also shown by the shift of the standard curve (see Fig. 2), meaning that the sensitivity of the of proEnk fragment 119-159 measurement is unexpectedly increased by one order of magnitude. Table 12: Comparison of single solid-phase antibody MTP assay compared to an assay using two different antibodies directed to two different non-overlapping regions of proEnk fragment 119- 159

[0224] EDTA-plasma samples were measured with three of the approaches as described above. The fold Signal to Reference (using AK1879 as solid phase antibody) was plotted for calibrators Controls, as well as EDTA-plasma samples (n=100) - Table 13 and Fig. 3, respectively.

[0225] Table 13: Fold Signal to Reference (AK1879) for single-solid phase antibodies (N-terminal AK3271 and C-terminal AK3278) compared to a combination of both.

[0226] 2.2.2. Dipstick immunochromatographic assay (bioaffinity immobilization of antibodies via streptavidin / biotin interaction)

[0227] A sandwich assay using two antibodies was used as reference. It comprised one biotinylated antibody against the C-terminal part of proEnk fragment 119-159 (AK1879) used for capturing and a second antibody coupled to gold nanoparticles against the mid-regional part of proEnk fragment 119-159 (AK2695) used for detection. Nitrocellulose membrane strips were coated with streptavidin (test line) to capture the sandwich complex formed during the reaction via its biotinylated antibody. For initiation of the reaction, buffer, antibodies, and sample were mixed using a reaction tube and one streptavidin coated membrane strip was added to the tube. The reaction mixture flowed through the strip for up to 10 minutes with the formed sandwich complex being captured at the test line (streptavidin). Afterwords the strip was dried at ambient condition and the formed colored test line, was measured for absorbance intensity using an appropriate reader.

[0228] If AK2260, which is directed against the N-terminal part of proEnk fragment 119-159, was added as a third „cold“ unlabeled antibody in the reaction mixture, the signals were only slightly increased compared to the reference assay (see Table 14 and Fig. 4). However, if adding biotinylated AK2260 as third antibody, thus using two biotinylated antibodies, one against the N- terminal part (AK2260) and one against the C-terminal part (AK1879) of proEnk fragment 119- 159, the signal more than doubled. This is also shown by the shift of the standard curve (see Fig. 4), meaning that the sensitivity of the of proEnk fragment 119-159 measurement is unexpectedly increased.

[0229] Table 14: Signals of reference antibody directed to C-terminal proEnk fragment 119-159 (AK1879) compared with addition of “cold” antibody directed to N-terminal proEnk 119-159 to the reaction mixture and use of both antibodies on the solid phase, respectively.

[0230] Figure Description

[0231] Fig- 1 - Dose / signal curves for proEnk fragment 119-159 for increasing concentrations of “cold” antibody against the N-terminal part of proEnk fragment 119-159 (AK 2259) added to the reaction mixture.

[0232] Fig- 2 - Standard curves for single solid-phase assays using either antibodies directed to the C- terminal part (AK 1879 and AK3278) or to the N-terminal part (AK3271) of proEnk fragment 119-159 compared to the standard curve for an assay using two solid-phase antibodies directed to both, the C-terminal (AK3278) and the N-terminal (AK3271) part of proEnk fragment 119-159, respectively.

[0233] Fig- 3 - Fold Signal to Reference (AK1879) for single-solid phase antibodies (N-terminal AK3271 and C-terminal AK3278) compared to a combination of both for calibrators, controls and EDTA- plasma samples, respectively.

[0234] Fig. 4 - Standard curves for dipstick immunochromatographic assays using C-terminal part of proEnk fragment 119-159 (AK1879) for capturing (biotin only as reference, and N-terminal proEnk fragment 119-159 antibody AK2260 as cold or second capturing antibody. Antibody against the mid-regional part of proEnk fragment 119-159 (AK2695) was coupled to gold nanoparticles and used as tracer (detection antibody).

Claims

CLAIMS1. Use of at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proenkephalin (proEnk) fragment 119-159 (SEQ ID No. 6) for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a sample of bodily fluid.

2. The use according to claim 1, wherein the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) is a specific determination.

3. The use according to claim 1 or 2, wherein said three different non-overlapping epitopes of proEnk fragment 119-159 are within the N-terminal region that is amino acid 119 to 133 (SEQ ID No. 2), within the mid-regional region that is amino acid 129 to 155 (SEQ ID No.7) and within the C-terminal region that is amino acid 152 to 159 (SEQ ID No. 5), wherein each of said epitopes comprises at least 4 or 5 amino acids.

4. The use according to claims 1 to 3, wherein said binder binding to the N-terminal region recognizes and binds to the N-terminal end (binds to amino acid 119, which is aspartic acid) of proEnk fragment 119-159 and wherein said binder binding to the C-terminal region recognizes and binds to the C-terminal end (binds to amino acid 159, which is serine) of proEnk fragment 119-159.

5. The use according to claims 1 to 4, wherein said binder is an antibody, an antibody fragment, an aptamer or a non-Ig-scaffold.

6. The use according to claims 1 to 5, wherein said at least three binders are utilized in an immunoassay.

7. The use according to claim 6, wherein said immunoassay is a sandwich immunoassay.

8. The use according to claims 1 to 7, wherein at least two of said binders directed to two different non-overlapping regions of proEnk fragment 119-159 are used as capture binders and at least one of said binders is directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 is labelled for detection.

9. The use according to claims 1 to 7, wherein at least two of said binders directed to two different non-overlapping regions of proEnk fragment 119-159 are labelled for detectionand at least one of said binders directed to a third region not overlapping with the first and second region of proEnk fragment 119-159 is a capture binder.

10. The use according to claims 1 to 7, wherein at least one of said binders directed to one region of proEnk fragment 119-159 is a capture binder, at least one of said binders directed to a second region of proEnk fragment 119-159 is labelled for detection and at least one of said binders directed to a third region of proEnk 119-159 is not labelled for detection and is not a capture binder, wherein said first, second and third region of proEnk fragment 119- 159 do not overlap.

11. The use according to claims 1 to 10, wherein said at least one capture binder is immobilized to a solid phase.

12. The use according to claim 11, wherein said solid phase is selected from the group of material comprising magnetic particles, paramagnetic particles, plastics (e.g. polystyrene, polypropylene, polycarbonate), cyclic olefin copolymers, glass, cellulose, nitrocellulose polyacrylamide, dextran, agarose, metal, or silicone.

13. The use according to claim 11, wherein said at least one capture binder is immobilized to said solid phase by non-covalent physical adsorption, bioaffinity immobilization techniques or covalent binding techniques.

14. The use according to claims 1 to 10, wherein said label is selected from the group comprising luminescent label, chemiluminescent label, electrochemiluminescence label, enzyme label, enzyme-amplified chemiluminescence label, fluorescence label, radioactive label, immunogold.

15. The use according to claim 1, wherein said bodily fluid is selected from the group comprising blood, cerebrospinal fluid, urine and saliva.

16. A kit for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) comprising at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6) in a sample of bodily fluid.

17. A method for the determination of the level of proEnk fragment 119-159 (SEQ ID No. 6) in a bodily fluid, the method comprising: - providing a sample of bodily fluid of a subject, determining the level of proEnk fragment 119-159 in said sample of bodily fluid, using at least three binders binding to three different non-overlapping epitopes comprised in the sequence of proEnk fragment 119-159 (SEQ ID No. 6).

Images