Title: Glucagon Assay

Field of the invention The invention is in the field of medical diagnostics and other medical tools, as well as uses thereof. More in particular, it provides antibodies specifically directed against glucagon. Even more in particular, the invention provides means and methods for detecting the presence and/or amount of glucagon in a sample. The invention also relates to specific peptide sequences of the epitope related to glucagon.

Background of the invention

Glucagon is a peptide hormone, produced by alpha cells of the pancreas. It works to raise the concentration of glucose and fat in the bloodstream, and is considered to be the main catabolic hormone of the body. It is also used as a medication to treat a number of health conditions. Its effect is opposite to that of insulin, which lowers the extracellular glucose.

The pancreas releases glucagon when the concentration of glucose in the bloodstream falls too low. Glucagon causes the liver to convert stored glycogen into glucose, which is released into the bloodstream. High blood- glucose levels, on the other hand, stimulate the release of insulin. Insulin allows glucose to be taken up and used by insulin-dependent tissues. Thus, glucagon and insulin are part of a feedback system that keeps blood glucose levels stable. Glucagon increases energy expenditure and is elevated under conditions of stress. Glucagon belongs to the secretin family of hormones.

Glucagon generally elevates the concentration of glucose in the blood by promoting gluconeogenesis and glycogenolysis. Glucagon also decreases fatty acid synthesis in adipose tissue and the liver, as well as promoting lipolysis in these tissues, which causes them to release fatty acids into circulation where they can be catabolised to generate energy in tissues such as skeletal muscle when required.

Glucose is stored in the liver in the form of the polysaccharide glycogen, which is a glucan (a polymer made up of glucose molecules). Liver cells (hepatocytes) have glucagon receptors. When glucagon binds to the glucagon receptors, the liver cells convert the glycogen into individual glucose molecules and release them into the bloodstream, in a process known as glycogenolysis. As these stores become depleted, glucagon then encourages the liver and kidney to synthesize additional glucose by gluconeogenesis. Glucagon turns off glycolysis in the liver, causing glycolytic intermediates to be shuttled to gluconeogenesis.

Glucagon also regulates the rate of glucose production through lipolysis. Glucagon induces lipolysis in humans under conditions of insulin suppression (such as diabetes mellitus type 1 ).

Glucagon production appears to be dependent on the central nervous system through pathways yet to be defined. In invertebrate animals, eyestalk removal has been reported to affect glucagon production. Excising the eyestalk in young crayfish produces glucagon-induced hyperglycemia.

Secretion of glucagon is stimulated by hypoglycemia, epinephrine (via b2, a2, and a1 adrenergic receptors), arginine, alanine (often from muscle- derived pyruvate/glutamate transamination, acetylcholine, and

cholecystokinin. On the other hand secretion of glucagon is inhibited by, somatostatin, insulin (via GABA), PPARy/retinoid X receptor heterodimer, increased free fatty acids and keto acids into the blood, and increased urea production. Abnormally elevated levels of glucagon may be caused by pancreatic tumors, such as glucagonoma, symptoms of which include necrolytic migratory erythema, reduced amino acids, and hyperglycemia, and may occur alone or in the context of multiple endocrine neoplasia type 1.

EP 0 111 216 relates to an enzyme immunoassay when a specific antibody produced by contacting a peptide essential to the formation of a specific antibody to a peptide antigen, resulting in a high reproducibility of the result of the enzyme immunoassay.

WO 2016/172631 relates to substrate selective inhibitors of insulin-degrading enzymes and uses thereof in e.g. pharmaceutical compositions.

EP 0 009 147 relates to novel peptide-enzyme conjugate obtained by coupling a labelling enzyme with a peptide useful in methods for enzyme immunoassay of pancreatic glucagon.

EP 2 100 901 relates to a method for protecting a peptide from pepti- dase activity in vivo, the peptide being composed of between 2 and 50 amino acids and having a C-terminus and an N-terminus and a C-terminus amino acid and an N-terminus amino acid is described.

GB 2 062 644 relates to a glucagon fragment, to a conjugated form of the fragment and a protein, to a process for preparation of a specific antibody using the conjugate and to a method of determining glucagon employing said antibody.

Moreover, glucagon is a hormone that helps manage the amount of glucose in the blood and as such is related to clinical conditions such as e.g. diabetes and obesity and thereto related conditions and complications. Present invention aims to solving the problem encountered when assaying glucagon and solves the problem with low specificity and/or high cross reactivity with other proteins.

Summary of the Invention

In one aspect, the invention relates to one or more amino acid sequences related to glucagon and as disclosed herein. Specifically, the invention relates to the amino acid sequence relating to the epitope of glucagon. Even more specifically, the invention relates to the neo-epitope of glucagon which is formed upon cleavage of oxyntomodulin to form glucagon.

In a further aspect, the invention also relates to antibodies which are capable specifically binding to the amino acid sequences of present invention. It is to be understood that the antibodies according to the invention specifically binds to the neo-epitope that are formed upon cleavage of oxyntomodulin to form glucagon. Thus, the antibodies according to the invention will not bind to uncleaved oxyntomodulin. Thus, the antibodies according to the invention will only specifically bind to the amino acid sequences as disclosed herein under the proviso that these sequences is the C-terminal of the cleaved antigen. Thus, the antigens to which an antibody according to the invention is capable to bind, is any of the sequences disclosed herein being the C-terminal in a peptide.

The antibody may be of any origin such as e.g. mammalian, such as e.g. human etc. The antibody may be monoclonal or polyclonal.

In a further aspect, the invention relates to an assay for quantification of glucagon in a biological sample. The quantification may be performed by employing the antibodies according to the invention. Measurement of the levels of glucagon in a blood sample may give

indications of several clinical conditions such as e.g. diabetes, glucagonoma (tumour of the pancreas) with symptoms of a skin rash called necrotizing migratory erythema, weight loss, mild diabetes, anaemia, stomatitis, glossitis, growth hormone deficiency in children, liver cirrhosis, low blood sugar (hypoglycemia), multiple endocrine neoplasia type I (disease in which one or more of the endocrine glands are overactive or form a tumour), pancreatitis (inflammation of the pancreas) etc.

High glucagon levels in the blood may contribute to the development of diabetes instead of just a low level of insulin.

The normal range of glucagon in the blood stream is in between about 50 to 100 pg/mL.

Consequently, the invention relates to a kit or a kit of parts comprising one or more antibodies according to the invention.

The invention also relates to use of antibodies according to the invention in any diagnostic or medical method.

The invention may relate to use of antibodies for diagnosing and/or treating diabetes, glucagonoma (tumour of the pancreas) with symptoms of a skin rash called necrotizing migratory erythema, weight loss, mild diabetes, anaemia, stomatitis, glossitis, growth hormone deficiency in children, liver cirrhosis, low blood sugar (hypoglycemia), multiple endocrine neoplasia type I (disease in which one or more of the endocrine glands are overactive or form a tumour), pancreatitis (inflammation of the pancreas) etc.

The invention also relates to use of one or more antibodies in any screening procedure. Such screening procedure may be screening of any clinical drug candidate capable of influencing the level of glucagon in the blood stream of a subject. One example may be drugs developed to decrease glucagon levels or block the signal from glucagon in the liver.

The invention also relates to a method of determining the levels of glucagon in a biological sample.

Description of Figures

Fig. 1 illustrates the Epitope mapping of the glucagon antibody show clear binding to peptide 6 in the ELISA set-up.

Fig. 2 illustrates the epitope mapping of the glucagon antibody show clear binding to the neoepitope exposed upon cleavage of oxyntomodulin to glucagon (peptides B06-B9) while peptides B10-B13 lacking the

-NT amino acids showed no binding.

Fig. 3 illustrates the epitope mapping of the glucagon antibody show clear binding to peptide 1 and peptide 6 in the RIA (radioimmunoassay).

Fig. 4 illustrates peptides B06 to B13 analyzed at 30pM in the RIA assay. Cross reactivity to oxyntomodulin (peptide 1 ) is only about 10% compared to peptide B06.

Detailed description of the invention

Present invention provides one or more of the following:

- An improved detection of glucagon in a sample,

- Decreased or eliminated cross-reactivity of an antibody according to the invention with other peptide sequences, such that decreased or eliminated cross-reactivity with oxyntomodulin is observed. In one aspect, present invention provides for an improved detection of glucagon in a sample and simultaneously a decreased or eliminated cross- reactivity of an antibody according to the invention with other peptide sequences, such that decreased or eliminated cross-reactivity with oxyntomodulin is observed.

The above technical problems are solved by the provision of the amino acid sequence, antibodies and methods disclosed herein. Particularly, SEQ ID NO.: 6 and 9 have been found to be useful in this respect. Moreover, sequences RAQDFVQWLMNT, and/or QDFVQWLMNT, and/or FVQWLMNT have also been found useful in the context of the invention and in solving the above mentioned technical problems. In one aspect, the invention relates to one or more amino acid sequences relating to glucagon. Specifically, the invention relates to an epitope of glucagon and in particular the neo-epitope formed upon cleavage of oxyntomodulin when this protein is cleaved to i.a. glucagon. The sequences of present invention comprises at least 6 amino acids, such as e.g. at least 7 amino acids, e.g. at least 8 amino acids, at least 9 amino acids, at least 10 amino acids. Preferably, the sequences of present invention comprise at least 10 amino acids. The one or more sequences relating to the invention may be one or more of any amino acid sequences comprising :

SEQ ID NO. 1 : 1. HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA or;

SEQ ID NO. 2: 2. HSQGTFTSDYSKYL

SEQ ID NO. 3: 3. FTSDYSKYLDSRR SEQ ID NO. 4: 4. YSKYLDSRRAQDF

SEQ ID NO. 5: 5. LDSRRAQDFVQWL

SEQ ID NO. 6: 6. RAQDFVQWLMNTR

SEQ ID NO. 7: 7. WLMNTKRNRNNIA

SEQ ID NO. 8: 8. KRNRNNIA or alternatively;

SEQ ID NO. 9: B06. RAQDFVQWLMNT

SEQ ID NO. 10 B07. QDFVQWLMNT

SEQ ID NO. 11 B08. FVQWLMNT

SEQ ID NO. 12 B09. QWLMNT

SEQ ID NO. 13 B10. QDFVQWLM

SEQ ID NO. 14 B11. RAQDFV

SEQ ID NO. 15 B12. RAQDFVQW

SEQ ID NO. 16 B13. RAQDFVQWLM

In one aspect, the invention relates to an amino acid sequence comprising RAQDFVQWLMNT. In a further aspect, present invention relates to an amino acid sequence having a sequence identity of at least about 70% to SEQ ID NO.: 9, such as e.g. at least about 75%, such as e.g. at least about 80%, such as e.g. at least about 85%, such as e.g. at least about 90%, such as e.g. at least about 95%, such as e.g. at least about 100% sequence identity to SEQ ID. NO.: 9. In yet a further aspect, present invention relates to an amino acid sequence having a sequence identity of at least about 90% to SEQ ID NO.: 9, such as e.g. at least about 95%, such as e.g.at least about 96%, such as e.g.at least about 97%, such as e.g. at least about 98%, such as e.g. at least about 99%, such as e.g. at least about 100% sequence identity to SEQ ID. NO.: 9. In another aspect, the invention relates to an amino acid sequence

comprising RAQDFVQWLMNTR. In a further aspect, present invention relates to an amino acid sequence having a sequence identity of at least about 70% to SEQ ID NO.: 6, such as e.g. at least about 75%, such as e.g. at least about 80%, such as e.g. at least about 85%, such as e.g. at least about 90%, such as e.g. at least about 95%, such as e.g. at least about 100% sequence identity to SEQ ID. NO.: 6.

In yet a further aspect, present invention relates to an amino acid sequence having a sequence identity of at least about 90% to SEQ ID NO.: 6, such as e.g. at least about 95%, such as e.g. at least about 96%, such as e.g. at least about 97%, such as e.g. at least about 98%, such as e.g. at least about 99%, such as e.g. at least about 100% sequence identity to SEQ ID. NO.: 6.

In a further aspect the invention relates to amino acid sequences

RAQDFVQWLMNT, and/or QDFVQWLMNT, and/or FVQWLMNT. In a further aspect, present invention relates to an amino acid sequence having a sequence identity of at least about 70% to RAQDFVQWLMNT, and/or QDFVQWLMNT, and/or FVQWLMNT, such as e.g. at least about 75%, such as e.g. at least about 80%, such as e.g. at least about 85%, such as e.g. at least about 90%, such as e.g. at least about 95%, such as e.g. at least about 100% sequence identity to RAQDFVQWLMNT, and/or QDFVQWLMNT, and/or FVQWLMNT.

In yet a further aspect, present invention relates to an amino acid sequence having a sequence identity of at least about 90% to RAQDFVQWLMNT, and/or QDFVQWLMNT, and/or FVQWLMNT, such as e.g. at least about 95%, such as e.g.at least about 96%, such as e.g. at least about 97%, such as e.g. at least about 98%, such as e.g. at least about 99%, such as e.g. at least about 100% sequence identity to RAQDFVQWLMNT, and/or

QDFVQWLMNT, and/or FVQWLMNT. In a further aspect, the invention relates to an amino acid sequence wherein the above mentioned sequences are the C-terminals of an amino acid sequence/peptide sequence.

Thus, the invention relates to any of the above mentioned amino acid sequences or a sequence with at least 70% sequence identity as set forth in the above sequences, such as e.g. such as e.g. at least about 75% sequence identity, such as e.g. at least about 80% sequence identity, such as e.g. at least about 85% sequence identity, such as e.g. at least about 90% sequence identity, such as e.g. at least about 95% sequence identity, such as e.g. at least about 98% sequence identity, such as e.g. at least about 99% sequence identity to any of the above mentioned sequences or an amino acid sequence identical to any of the above mentioned sequences.

In one aspect, the invention relates to use of any of the above amino acid sequences for detection and/or determination and/or quantification of glucagon. The detection and/or determination and/or quantification of glucagon may be in any sample, such as e.g. a biological sample.

Specifically, the invention relates to use of an amino acid comprising or consisting of RAQDFVQWLMNT for detection and/or determination and/or quantification of glucagon in a sample.

Moreover, the invention relates to use of an amino acid comprising or consisting of RAQDFVQWLMNTR for detection and/or determination and/or quantification of glucagon in a sample.

Furthermore, the invention relates to use of an amino acid comprising or consisting of RAQDFVQWLMNT, and/or QDFVQWLMNT, and/or

FVQWLMNT for detection and/or determination and/or quantification of glucagon in a sample. Moreover, present invention also relates to an antibody capable of specifically binding to one or more of any amino acid sequences comprising: RAQDFVQWLMNTR

RAQDFVQWLMNT QDFVQWLMNT FVQWLMNT QWLMNT

It is to be understood that the invention relates to an antibody capable of specifically binding to the neo-epitopes as discussed herein. Consequently, in one aspect, the antibodies according to the invention specifically binds to the above mentioned amino acid sequences, wherein such amino acid sequence may be the C-terminal of an amino acid sequence. Put in another wording, the antibodies according to the invention are capable of binding to and recognising the peptide sequence as disclosed herein, where such sequences are part of an entire antigen. The invention may thus also in addition to the antibodies specifically capable of binding to or recognising the peptide sequences as disclosed herein, further comprise antibodies that are capable of binding to the same antigen but specifically not binding to any one of the peptide sequences as disclosed herein. Specifically, the invention relates to an antibody capable of specifically binding to an amino acid comprising or consisting of the sequence

RAQDFVQWLMNT.

Moreover, the invention relates to an antibody capable of specifically binding to an amino acid comprising or consisting of the sequence

RAQDFVQWLMNTR. Furthermore, the invention relates to an antibody capable of specifically binding to an amino acid comprising or consisting of the sequences

RAQDFVQWLMNT, and/or QDFVQWLMNT, and/or FVQWLMNT.

The antibody may be of any origin such as e.g. any type of animal and specifically of mammalian origin. Non-limiting examples of the origin of the antibodies according to the invention may be e.g. rabbit, rat, goat or of any avian origin or laternatively or any insect origin. The origin of the antibody may be of human origin. The antibody may be polyclonal or monoclonal. Preferably, the antibody is monoclonal and even more preferably a

monoclonal human antibody. It is to be understood that antibodies according to the invention may be derivable from natural sources, but further

manipulated so as provide for antibodies that are not naturally occurring.

In another aspect, the antibodies according to the invention are

artificial/synthetic, such as e.g. artificial monoclonal antibodies and notably recombinant antibodies or chimeric antibodies. Alternatively, the antibodies according to the invention may be artificial/synthetic polyclonal antibodies. In a further aspect, the agents capable of recognising the peptide sequences disclosed herein are e.g. antibody mimics that may be nucleic acids and small molecules or antibody fragments and fusion proteins composed from these. In yet a further aspect, the agent may be an aptamer. The invention also relates to use of an antibody according to the invention in an assay. In principle, the assay may be any type of assay known in the art. One type of assay may be an ELISA (Enzyme-Linked Immunosorbent Assay) type of assay. The Elisa assay may be of any type known in the art such as e.g. direct ELISA, indirect ELISA, sandwich ELISA, or competitive ELISA. Another example of an assay is radioimmunoassay (RIA). The antibody may optionally be conjugated with a suitable enzyme, such as e.g. Horse Radish Peroxidase (HRP), alkaline phosphatase (ALP), urease or any other suitable enzyme known in the art. This aspect of the invention is suitable for e.g. direct ELISA or competitive ELISA.

Radioimmunoassay (RIA)

In one aspect, the invention relates to an antibody for use in a RIA. The antibody is capable of binding to any of the amino acid sequences as disclosed herein. In such assay, the assay may further comprise a

radioactively labelled target to which the antibody is capable of binding. In one aspect the target may be radioactively labelled glucagon. Moreover, in the use of the antibody, the glucagon present in the patient sample will competitively bind to the antibody of the invention. Antibody-bound glucagone is separated from the un-bound fraction and radioactivity is measured in the bound fraction. It is to be understood that the glucagon in the patient sample is the neo-epitope relating to any one of SEQ ID NO.: 6 and SEQ ID NO.: 9-12. The antibody may or may not be immobilised on a solid support such as the wall of a well or any surface.

For example, the antibody of present invention may be mixed with the radiolabelled target such as e.g. radiolabelled glucagon. The available binding sites of the antibody will thus be saturated with bound radiolabelled target. Upon contacting the antibody bound to the radiolabelled target with a biological sample, the non-radiolabelled target competitively binds to the antibody after which measurement of the released radiolabelled target is performed, allowing for a quantitative measurement.

Direct ELISA

In one aspect, the invention relates to the antibody according to the invention is conjugated with a suitable enzyme as described herein. Such antibody may be denoted as a primary antibody. The mode of action is such that the primary antibody binds to one or more sequences (the antigen) according to the invention, i.e. binds to one or more of amino acid sequences SEQ ID NO.: 6 and SEQ ID NO.: 9-12. Once the antibody is bound to an antigen, a substrate is added which is digestible by the enzyme conjugated to the primary antibody. This in turn allows for quantification.

The enzyme conjugated to the antibody may be any suitable enzyme known in the art such as e.g. Horse Radish Peroxidase (HRP), alkaline phosphatase (ALP), urease etc.

The substrate may be any suitable substrate which is capable of being digested by the conjugated enzyme. Non-limiting examples of such substrates are e.g. tetramethyl benzidine (TMB), 2,2'-Azinobis [3- ethylbenzothiazoline-6-sulfonic acidj-diammonium salt (ABTS), p-Nitrophenyl Phosphate, Disodium Salt (PNPP), o-phenylenediamine dihydrochloride (OPD) etc.

Indirect ELISA

In a further aspect, the antibody according to the invention is not conjugated with a suitable enzyme as described herein. Such antibody may be denoted as a primary antibody. The primary antibody is capable of binding to one or more of amino acid sequences SEQ ID NO.: 6 and SEQ ID NO.: 9-12.

In such instance the primary antibody is not conjugated with an enzyme, the invention further comprises an antibody capable of recognising the Fc part of the primary antibody. Such antibody is denoted as a secondary antibody. The secondary antibody may optionally be conjugated with a suitable enzyme, such as e.g. e.g. Horse Radish Peroxidase (HRP), alkaline phosphatase (ALP), urease or any other suitable enzyme known in the art. In order to allow for detection and ultimately quantification, a substrate capable of being digested by the enzyme conjugated to the secondary antibody is added. The substrate may be any suitable substrate which is capable of being digested by the conjugated enzyme. Non-limiting examples of such substrates are e.g. tetramethyl benzidine (TMB), 2,2'-Azinobis [3- ethylbenzothiazoline-6-sulfonic acidj-diammonium salt (ABTS), p-Nitrophenyl Phosphate, Disodium Salt (PNPP), o-phenylenediamine dihydrochloride (OPD) etc.

Sandwich ELISA

The invention also relates to a capture antibody capable of specifically binding to glucagon at a site different from the neo-epitope described herein (i.e. sites having sequences different from amino acid sequences SEQ ID NO.: 6 and SEQ ID NO.: 9-12). Consequently, the capture antibody is different from the antibody according to the invention which is capable of binding to one or more of amino acid sequences SEQ ID NO.: 6 and SEQ ID NO.: 9-12 and consequently binds to a different epitope of e.g. glucagon. In this context, the antibody according to the invention capable of binding to any one of SEQ ID NO.: 6 and SEQ ID NO.: 9-12 is denoted as the detection antibody. The detection antibody is enzyme linked with a suitable enzyme, such as e.g. e.g. Horse Radish Peroxidase (HRP), alkaline phosphatase (ALP), urease or any other suitable enzyme known in the art.

In order to allow for detection and ultimately quantification, a substrate capable of being digested by the conjugated enzyme is added. The substrate may be any suitable substrate which is capable of being digested by the conjugated enzyme. Non-limiting examples of such substrates are e.g.

tetramethyl benzidine (TMB), 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acidj-diammonium salt (ABTS), p-Nitrophenyl Phosphate, Disodium Salt (PNPP), o-phenylenediamine dihydrochloride (OPD) etc. Competitive ELISA

In one aspect, the invention relates to an antibody capable of binding to the antigen of a biological sample, wherein the antigen comprises any one of sequences SEQ ID NO.: 6, and/or SEQ ID NO.: 9-12. This antibody may be denoted as the primary antibody.

In this aspect the invention further relates to an antibody capable of binding to the Fc part of the primary antibody. This antibody may thus be suitable denoted as the secondary antibody. The secondary antibody may optionally be conjugated with a suitable enzyme, such as e.g. e.g. Horse Radish

Peroxidase (HRP), alkaline phosphatase (ALP), urease or any other suitable enzyme known in the art.

In order to allow for detection and ultimately quantification, a substrate capable of being digested by the conjugated enzyme is added. The substrate may be any suitable substrate which is capable of being digested by the conjugated enzyme. Non-limiting examples of such substrates are e.g.

tetramethyl benzidine (TMB), 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acidj-diammonium salt (ABTS), p-Nitrophenyl Phosphate, Disodium Salt (PNPP), o-phenylenediamine dihydrochloride (OPD) etc.

In order to permit detection and quantification, the primary antibody is incubated with the antigen from a biological sample. The primary antibody bound to its antigen from the biological sample is then added to a surface which may typically by the surface of any type of container. The surface is pre-coated with the same antigen. Any unbound primary antibody (i.e. not bound to said surface), is typically removed by any suitable method such as e.g. rinsing. Said surface is then contacted with the secondary antibody, which then binds to the Fc part of the primary antibody bound to said surface. A suitable substrate is then added which upon digestion of the enzyme conjugated to the secondary antibody allows for detection and quantification. Overall and as mentioned herein, the relevant antibodies are conjugated to a suitable enzyme. Suitable enzymes are known in the art and exemplified herein. In performing the assays mentioned herein, a suitable substrate is added for the purpose of detection and quantification. Several suitable substrates are known in the art and consequently, the methods and kits described herein may or may not comprise a substrate which, upon digestion, results in a chromogenic or fluorescent signal.

Present invention also relates to a kit or a kit of parts. The kit may comprise a one or more antibodies as described herein optionally conjugated with a suitable enzyme, such as e.g. e.g. Horse Radish Peroxidase (HRP), alkaline phosphatase (ALP), urease or any other suitable enzyme known in the art. The one or more antibodies may be the primary or detection antibodies as described herein. These antibodies may optionally be conjugated to a suitable enzyme.

The kit may further comprise a secondary antibody as described herein optionally conjugated with a suitable enzyme, such as e.g. e.g. Horse Radish Peroxidase (HRP), alkaline phosphatase (ALP), urease or any other suitable enzyme known in the art.

The kit may further comprise a capture antibody capable of specifically binding to glucagon at the neo-epitope described herein (i.e. sites from amino acid sequences SEQ ID NO.: 6 and SEQ ID NO.:9-12). In some aspects, the capture antibody is in solution. In a preferred aspect, the capture antibody is directly or indirectly attached to a surface of any kind. For example, the capture antibody may be attached to the surface of a well, such as e.g. the well of a microtiter plate or any format. The kit may further comprise a suitable substrate. The substrate may be any substrate capable of being digested by the enzyme conjugated to the relevant antibody as the case may be.

The kit may further comprise a control and/or internal standard.

The kit may be in any suitable form, such as comprising one or more containers. For example, the kit may comprise a microtiter plate of any suitable format. The one or more containers may be coated with the capture antibodies according to the invention on one or more surfaces of the one or more containers. In one aspect, the containers may also be coated with the antigen capable of being recognised by the relevant (primary) antibody.

Specifically, a kit or kit of parts according to the invention may be designed to suite the desired context for the assay and its methodology.

Kit for R! A

In one aspect the kit according to the invention may comprise:

a) an antibody according to the invention capable of binding an antigen comprising any one of SEQ ID NO.: 6 and SEQ ID NO.: 9-12

b) radiolabelled antigen to which the antibody according to a) is capable of binding.

Kit for Direct ELISA

In a further aspect of the invention, the kit according to the invention may comprise:

a) an antibody according to the invention capable of binding an antigen comprising any one of SEQ ID NO.: 6 and SEQ ID NO.: 9-12, optionally further conjugated to an enzyme

b) optionally a substrate which is digestible by the enzyme in a). Kit for Indirect ELISA

In one aspect of the invention, the kit according to the invention may comprise:

a) an antibody according to the invention capable of binding an antigen comprising any one of SEQ ID NO.: 6 and SEQ ID NO.:9-12,

b) an antibody optionally conjugated to an enzyme, capable of binding to the Fc-part of the antibody in a),

c) optionally a substrate which is digestible by the enzyme in b). Kit for Sandwich ELISA

In a further aspect of the invention, the kit according to the invention may comprise:

a) an antibody according to the invention capable of binding an antigen comprising any one of SEQ ID NO.: 6 and SEQ ID NO.:9-12,

b) an antibody capable of binding to the same antigen in a), but not binding to any one of SEQ ID NO.: 6 and SEQ ID NO.:9-12,

c) an antibody optionally conjugated to an enzyme capable of binding to the Fc part of the antibody in a),

d) optionally a substrate which is digestible by the enzyme in c).

Kit for Competitive ELISA

In yet a further aspect of the invention, the kit according to the invention may comprise:

a) an antibody according to the invention capable of binding an antigen comprising any one of SEQ ID NO.: 6 and SEQ ID NO.:9-12,

b) a surface pre-coated with the antigen in a),

c) an antibody optionally conjugated to an enzyme capable of binding to the Fc part of the antibody in a),

d) optionally a substrate which is digestible by the enzyme in c).

RIA Present invention also relates to a method of quantifying the presence of an antigen. The antigen may in principle be any antigen such as e.g. glucagon in a biological sample. Consequently, a method according to the invention may comprise:

a) providing an antibody according to the invention, wherein said antibody is pre-loaded with radiolabelled antigen

b) contacting the antibody bound to radiolabelled antigen in a) with a patient sample,

c) measuring the released radiolabelled antigen

Direct ELISA

In one aspect of the invention, the method may comprise:

a) providing a biological sample from a subject,

b) contacting the sample with the primary antibody according to the invention c) adding to the sample a suitable substrate, and

d) measuring the output of the digested substrate.

The output may be a chromogenic or fluorescent signal.

Indirect ELISA

Alternatively, the method may comprise:

a) providing a biological sample from a subject,

b) contacting the sample with the primary antibody according to the invention, c) adding to the sample a secondary antibody according to the invention, d) adding to the sample a suitable substrate, and

e) measuring the output of the digested substrate. The output may be a chromogenic or fluorescent signal. Sandwich ELISA

A further alternative, the method according to the invention may comprise the steps of:

a) providing a biological sample from a subject,

b) contacting the sample with the capture antibody according to the invention, c) adding to the sample a detection antibody according to the invention, d) adding to the sample a secondary antibody according to the invention, e) adding to the sample a suitable substrate, and

f) measuring the output of the digested substrate.

The output may be a chromogenic or fluorescent signal.

Competitive ELISA

In yet a further alternative, the method according to the invention may comprise the steps of:

a) Contacting the primary antibody according to the invention with a biological sample containing an antigen,

b) contacting the primary antibody-antigen complex from step a) with a surface which is pre-coated with the same antigen as in a),

c) removing any unbound antibody in step b),

d) adding a secondary antibody to said surface,

e) adding a substrate, and

f) measuring the output of the digested substrate. The output may be a chromogenic or fluorescent signal.

It is to be understood that the methods described above may comprise rinsing steps in between any of the steps in the method. For example, a rinsing step may suitably take place between e.g. step b) and c) or c) and d). It is also to be understood that the invention may comprise any substrate enabling any form of detection known in the art. For example, the substrate may be used for detection by absorbance or fluorescence or by

electrochemical signal in order to quantify the amount of glucagon in a sample. Thus measuring the output in the method as disclosed herein relates to recording the absorbance or fluorescence or electrochemical signal after the substrate has been digested by the enzyme. The measurement may further we compared of normed or put in relation to an internal control or standard.

The methods and the kits as described above may further comprise the use of a suitable control and/or internal standard.

The method according to the invention may comprise the use of the kit according to the invention for detection and/or quantification of e.g. glucagon in a sample.

The invention is further illustrated in the below seen non-limiting examples.

EXAMPLES

Eight peptides covering the full sequence of glucagon (sequence 1 , excluding the bold/underlined amino acids) and oxyntomodulin (sequence 1 , including the bold/underlined amino acids) was ordered from Schafer-N in Denmark in November 2016. All peptides were biotin-tagged:

1. Biotin-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA

2. Biotin-HSQGTFTSDYSKYL

3. Biotin-FTSDYSKYLDSRR

4. Biotin-YSKYLDSRRAQDF

5. Biotin-LDSRRAQDFVQWL 6. Biotin-RAQDFVQWLM NTKR (one aa excluded to make it possible to synthesis)

7. Biotin-WLMNTKRNRNNIA

8. Biotin-KRNRNNIA

For fine tuning of the identified epitope (no 6) above, a second round of peptides were ordered from Schafer-N. These eight (8) peptides covered only peptide no 6 from the first test as this was identified to contain the binding epitope. All peptides were biotin-tagged:

B06 Biotin-RAQDFVQWLMNT

B07 Biotin-QDFVQWLMNT

B08 Biotin-FVQWLMNT

B09 Biotin-QWLMNT

B10 Biotin-QDFVQWLM

B11 Biotin-RAQDFV

B12 Biotin-RAQDFVQW

B13 Biotin-RAQDFVQWLM

ELISA

In the ELISA set-up the glucagon antibody was incubated in solution

(dilutions 1/1000-1/128 000) with the individual peptides (5ug/mL) before adding them to a streptavidin plate. The peptide-antibody complexes bound to the plate were detected by addition of an AP-conjugated secondary antibody (swine anti rabbit-AP, dilution 1/1000).

RIA

High concentrations (1000 pM) of peptides 1-8 were analyzed as samples in the RIA using the ordinary assay protocol. The high concentration of peptide (20 times higher than the calibrator, which is 0-150 pM) was used as most of the peptides were not expected to give a signal. The second set of peptides (B06-B13) were analyzed at a lower concentration 30 pM which is within the assay range.

Results and discussions

ELISA

In the first ELISA test a clear binding was seen by the glucagon antibody to peptide 6. There was also a limited binding to peptide 1 (oxyntomodulin) which is less desirable (figure 1 ). On one hand this it is not surprising as oxyntomodulin contains the exact sequence of peptide 6 but earlier data, reported in the IFU, show no cross reactivity of the glucagon antibody to oxyntomodulin. To clarify the binding epitope, a new set of peptides covering peptide 6 was ordered. These peptides contain different sequences of peptide 6 and four of them contain the neoepitope exposed when

oxyntomodulin is cleaved to glucagon (B06-B09 above).

Analyzing the second set of peptides (B06-B13) in the ELISA confirmed the binding of the glucagon antibody to peptide 6. It also gave additional information that the neoepitope exposed upon cleavage of oxyntomodulin to glucagon is essential for the binding as the peptides containing this epitope (B06-B09) showed good binding while the peptides B10-B13 lacking the -NT amino acids did not show any binding at all (figure 2). The three-dimensional structure of the peptide may also be important as binding to the neoepitope was reduced when the peptide contained less than 10 amino acids (B08-B09, figure 2).

RIA

The first set of peptides gave a similar result in the RIA as in the ELISA with clear binding to peptide 6. However, the unwanted cross reactivity to oxyntomodulin (peptide 1 ) was more pronounced than in the ELISA. This could be due to the very high peptide concentrations used (1000pM). There was no or very low binding to the other peptides (figure 3).

Analyzing the second set of peptides in the RIA a very clear result could be seen when peptides were analyzed at a concentration within the normal assay range, 30pM (figure 4). Here the binding to peptide B06 was much higher than to any of the other peptides. Remarkable was that when one amino acid was removed between peptide6 and peptide B06 exposing the neoepitope of glucagon, binding was increased 7-fold.

When the sequence is shortened with two or more amino acids (B07-B09) the binding is markedly decreased or altogether lost. It should also be noted that at 30pM the cross reactivity to oxyntomodulin (peptide 1 ) is only about 12% (figure 4).

Itemln specific embodiments, present invention relates to the following items:

1. An amino acid sequence having at least 70% sequence identity to any one of SEQ ID. NO.: 6 and 9-12

2. The amino acid sequence according to any of the preceding items, wherein the amino acids is any one of SEQ ID NO.: 6.

3. The amino acid sequence according to any of the preceding items, wherein the amino acids is any one of SEQ ID NO.: 9.

4. An antibody capable of binding to any one of the amino acids sequences as set forth in any of the preceding items.

5. Use of an antibody according to item 4 for detection and/or quantification of glucagon in a biological sample. 6. A kit, wherein the kit comprises

a) an antibody according to item 4, and optionally further comprising:

b) a radiolabelled antigen to which the antibody according to a) is capable of binding.

7. A kit, wherein the kit comprises

a) an antibody according to item 4, optionally further conjugated to an enzyme

b) optionally a substrate which is digestible by the enzyme in a).

8. A kit, wherein the kit comprises

a) an antibody according to item 4,

b) an antibody optionally conjugated to an enzyme, capable of binding to the Fc-part of the antibody in a),

c) optionally a substrate which is digestible by the enzyme in b).

9. A kit, wherein the kit comprises

a) an antibody according to item 4,

b) an antibody capable of binding to the same antigen in a), but not binding to any one of SEQ ID NO.: 6 and SEQ ID NO.: 9-12

c) an antibody optionally conjugated to an enzyme capable of binding to the Fc part of the antibody in a),

d) optionally a substrate which is digestible by the enzyme in c). 10. A kit, wherein the kit comprises

a) an antibody according to item 4,

b) a surface pre-coated with the antigen to which the antibody in a) can bind, c) an antibody optionally conjugated to an enzyme capable of binding to the Fc part of the antibody in a),

d) optionally a substrate which is digestible by the enzyme in c). 11. A method for detection and/or quantification of an antigen, the method comprising the steps of:

a) providing an antibody according to item 4, wherein said antibody is pre- loaded with radiolabelled antigen,

b) contacting the antibody bound to radiolabelled antigen in a) with a patient sample,

c) measuring antigen bound to the radiolabelled antigen.

12. A method for detection and/or quantification of an antigen, the method comprising the steps of:

a) providing a biological sample from a subject,

b) contacting the sample with the primary antibody according to item 4, c) adding to the sample a suitable substrate, and

d) measuring the output of the digested substrate.

13. A method for detection and/or quantification of an antigen, the method comprising the steps of:

a) providing a biological sample from a subject,

b) contacting the sample with the primary antibody according to item 4, c) adding to the sample a secondary antibody according to the invention, wherein the secondary antibody is capable of binding to the Fc part of the antibody in b),

d) adding to the sample a suitable substrate, and

e) measuring the output of the digested substrate.

14. A method for detection and/or quantification of an antigen, the method comprising the steps of:

a) providing a biological sample from a subject,

b) contacting the sample with the capture antibody according to item 4 capable of binding to the antigen in the biological sample,

c) adding to the sample a primary antibody capable of binding to the antigen, d) adding to the sample a secondary antibody capable of binding to the Fc part of the antibody in c), wherein the secondary antibody is conjugated to an enzyme,

e) adding to the sample a suitable substrate, and

f) measuring the output of the digested substrate,

wherein the antibody in b) specifically binds to an epitope different from the antibody in c).

15. A method for detection and/or quantification of an antigen, the method comprising the steps of:

a) contacting an antibody according to item 4 with a biological sample containing an antigen,

b) contacting the antibody-antigen complex from step a) with a surface which is pre-coated with the same antigen as in a),

c) removing any surface unbound antibody in step b),

d) adding a secondary antibody to said surface,

e) adding a substrate, and

f) measuring the output of the digested substrate.