FIELD OF THE INVENTION

The invention relates to a composition, to a use, to a method for purifying at least one peptide or protein and to a method for quantifying gluten.

BACKGROUND

Prolamins are gluten proteins rich in proline, and in addition to their high proline content they are also rich in glutamine. Prolamins are characterized by their water- insolubility, but instead they are typically soluble in aqueous alcohol. The prolamins of three cereals, i.e. wheat, rye and barley, which are also called Triticeae cereals, are considered to contain sequences harmful to those having gluten intolerance or celiac disease.

Celiac disease, also referred to as gluten enteropathy or non-tropical sprue, is an autoimmune disorder of the small intestine that occurs in genetically predisposed people of all ages. The characteristic symptom of this disease is the damage of the epithelium of small bowel and intolerance to wheat glu ^¬ ten. Products containing wheat proteins as well as barley and rye should be eliminated from the diet of people suffering from this disease. Assays for the detection and quantitation of glu ^¬ ten in foodstuffs may be required for determining whether the foodstuffs are gluten-free.

The current gluten quantification method based on R5 antibody, recommended by the EU Codex Alimentarius standard (118-1979), with appropriate extraction, performs well in most food matrices. In quantification of hordein in barley-containing foods, however, it leads to overestimation . One reason for this may be that the current reference material is a selection of 28 European wheat cultivars. There is thus a need for improved ref- erence materials for gluten detection and quantification.

SUMMARY A composition comprising at least one purified peptide or protein is disclosed, wherein the at least one peptide or protein comprises at least one amino acid stretch having the se- quence QQPFP (SEQ ID NO: 1) .

Use of at least one purified peptide or protein or of the composition as a reference material for quantifying the amount of gluten present in a food sample is also disclosed.

A method for purifying at least one peptide or protein from a starting material is further disclosed, comprising extracting at least a portion of the at least one peptide or pro ^¬ tein present in the starting material with an aqueous solution comprising an alcohol; and purifying the at least one peptide or protein from the aqueous solution to obtain a composition com- prising the at least one peptide or protein, wherein the at least one peptide or protein comprises at least one amino acid stretch having the sequence QQPFP (SEQ ID NO: 1) .

A method for quantifying the amount of gluten present in a food sample is disclosed, the method comprising extracting at least a portion of the gluten from the food sample, contact ^¬ ing the extracted gluten with an antibody, thereby detecting the amount of the gluten; wherein the method comprises quantifying the amount of gluten in the food sample using the composition or the at least one purified peptide or protein as a reference ma- terial.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are included to provide a further understanding of the invention and constitute a part of this specification, illustrate embodiments and together with the description help to explain the principles of the invention. In the figures:

Figure 1 is a reverse-phase liquid chromatogram of to ^¬ tal hordein extract separated on a C8 column;

Figure 2 shows the reaction of isolated hordeins against R5 antibody in sandwich ELISA; Figure 3 (a) is a cation-exchange chromatogram of hor- dein and (b) is a reverse-phase chromatogram of purified C- hordein and whole hordein from cv. Vorma' ;

Figure 4 shows the reaction of C-hordein mixed with bo ^¬ vine serum albumin (BSA) in different ratios against R5 antibody (a) in sandwich ELISA; and (b) in competitive ELISA. Figure 4 (c) illustrates the reaction of purified whole hordein of 6 cul- tivars in R5 sandwich ELISA compared with 30%, 40% and 50% C- hordein standards and gliadin standard; and

Figure 5 shows the prolamin concentration of gluten- free oat flour spiked with three barley flours, determined by HPLC, R5 sandwich ELISA with 40% C-hordein standard, and R5 sandwich ELISA with gliadin standard.

DETAILED DESCRIPTION

The embodiments of the invention described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined together to form a further embodi- ment . A method, a composition or a use, to which the invention is related, may comprise at least one of the embodiments de ^¬ scribed hereinbefore.

The present inventors have found that the proportion of particular peptides and/or proteins in gluten-containing materi- als, such as the proportion of C-hordein in whole barley horde- in, may explain the hordein overestimation with gliadin reference material in methods for gluten quantification, such as R5 antibody-based enzyme-linked immunosorbent assay (ELISA) or oth ^¬ er R5 antibody-based assays. Further, particular purified pep- tides or proteins may serve as a reference material for quanti ^¬ fying gluten and components thereof, such as hordein, for example, to determine the presence of gluten, such as barley contam ^¬ ination in oats. The presence of certain amino acid stretches in the peptides or proteins may ensure efficient detection using an antibody assay, such as ELISA using the R5 antibody.

The antibody "R5" refers to the antibody described in Sorell et al . , 1998, FEBS Lett. 439, 46-50. In the context of this specification, the term "reference material" may, in an embodiment, refer to a standard and/or to a calibrant. A standard may be considered to be a substance that is established, for example by an authority, as a rule for the measure of quantity and optionally of weight or quality of said substance or, in this specification of gluten, in a test sample. A calibrant may be considered to be a substance that is used to calibrate an analytical method, for example an analyti- cal method for measuring the quantity and optionally of weight or quality of said substance or, in this specification of glu ^¬ ten, in a test sample. The reference material, standard and/or calibrant may comprise, but does not necessarily comprise, addi ^¬ tional proteins or peptides in addition to the at least one pu- rified peptide or protein.

A composition comprising at least one purified peptide or protein is disclosed, wherein the at least one peptide or protein comprises at least one amino acid stretch having the se ^¬ quence QQPFP (SEQ ID NO: 1) .

In the context of this specification, the term "at least one peptide or protein" may be understood as referring to one, two or a plurality of peptides and/or proteins. The term "peptide" may be understood as referring to polypeptides as well as smaller peptides, such as a peptide comprising or consisting of one, two or more of the amino acid stretches. The at least one peptide or protein may, for instance, refer to a prolamin fraction or to a heterogeneous group of peptides and/or pro ^¬ teins. The at least one peptide or protein may comprise or con ^¬ sist of at least one prolamin protein. It may comprise or con- sist of at least one synthetic or recombinant peptide or pro ^¬ tein, or it may comprise or be at least one peptide or protein extracted from cereal material. It may be, but is not particu ^¬ larly limited to, at least one monomeric protein. The at least one protein may be native, i.e. in a native form. The at least one peptide or protein may be essentially soluble in an aqueous solution comprising an alcohol.

In the context of this specification, the term "pep ^¬ tide" may refer to a peptide of any size, provided it comprises at least one amino acid stretch having the sequence QQPFP (SEQ ID NO: 1), and may also refer to a polypeptide. Thus the term "at least one peptide or protein" may refer to peptides and/or proteins, i.e. amino acid chains, of any size.

In an embodiment, the at least one peptide or protein is sulphur poor. In the context of this specification, the term "sulphur poor" may refer to at least one peptide or protein that contains less than 1 mol-%, or less than 0.5 mol-% of cysteine residues. It may alternatively or additionally refer to at least one peptide or protein that contains at most 5, or at most 4, or at most 3, or at most 2, or at most 1 cysteine residue ( s ) /mol , or no cysteine residues.

In the context of this specification, the term "amino acid stretch" may be understood as referring to a contiguous amino acid sequence in the at least one peptide or protein. As the peptide or protein may comprise a plurality of the amino ac ^¬ id stretches, they may also be referred to as repeats.

The at least one peptide or protein may comprise a plu- rality of amino acid stretches having the sequence QQPFP (SEQ ID NO: 1) .

The at least one peptide or protein may comprise at least one amino acid stretch having the sequence PQQPFPQQ (SEQ ID NO: 2) .

The at least one peptide or protein may comprise a plu ^¬ rality of amino acid stretches having the sequence PQQPFPQQ (SEQ ID NO: 2) .

The at least one peptide or protein may comprise at least 5, at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13 amino acid stretches having the sequence QQPFP (SEQ ID NO:

1) .

The at least one peptide or protein may comprise at least 5, at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13 amino acid stretches having the sequence PQQPFPQQ (SEQ ID NO:

2) . The at least one peptide or protein may comprise or be at least one prolamin protein. The at least one peptide or pro ^¬ tein may be at least one native prolamin protein. Native prola- min proteins are obtainable from barley, wheat or rye grains us ^¬ ing a suitable method, such as the method for purifying at least one peptide or protein according to one or more embodiments de ^¬ scribed in this specification. In an embodiment, the at least one prolamin protein is sulphur poor.

The at least one peptide or protein may comprise or be at least one prolamin protein selected from the group consisting of C-hordein, ω-gliadin, ω-secalin and any combinations or mixtures thereof.

In an embodiment, the at least peptide or protein com- prises or is C-hordein. C-hordein is relatively readily obtaina ^¬ ble from barley grains .

S-poor prolamins, such as C-hordein of barley, ω- gliadin of wheat and ω-secalin of rye, typically consist essen ^¬ tially or mainly of the repeats, i.e. amino acid stretches, PQQPFPQQ (SEQ ID NO: 2) and non-repetitive sequences at the isl ^¬ and C-termini.

However, the presence of non-repetitive sequences with ^¬ in the at least one peptide or protein and/or at the N- and C- terminals of the at least one peptide or protein are not exclud- ed.

In an embodiment, the composition is obtainable by the method for purifying the at least one peptide or protein accord ^¬ ing to one or more embodiments described in this specification.

The composition may comprise at most 5 w-%, or at most 3 w-%, or at most 2 w-%, or at most 1 w-% of gluten proteins other than the at least one peptide or protein by weight of the total dry protein in the composition.

The expression "gluten proteins" or any corresponding expression should be understood in this specification, unless otherwise stated, as proteins found in cereal that are consid ^¬ ered to be harmful to a person with celiac disease and/or an in ^¬ tolerance to gluten. In an embodiment, the gluten proteins are cereal prolamins and/or glutelins found in the Triticeae cere- als, i.e. in wheat, rye and barley. The prolamins of these cere ^¬ als in particular are considered to contain protein sequences harmful to those having gluten intolerance or celiac disease. These prolamins include gliadin, which can be found in wheat, hordein which can be found in barley, and secalin, which can be found in rye. Prolamins may be defined as the storage proteins in the endosperm of cereal grains that are soluble in aqueous alcohol solution, but insoluble in water and 0.5 M NaCl in their native form. As the one or more peptides or proteins may also be considered gluten proteins, the gluten proteins may be gluten proteins other than the at least one peptide or protein accord ^¬ ing to one or more embodiments described in this specification. For instance, the gluten proteins other than the at least one peptide or protein may include hordeins other than C-hordeins, e.g. D-hordeins, γ-hordeins, B-hordeins and/or correspondings prolamins in other cereals, such as HMW-glutenin subunits x- and y-types, γ-gliadins, /β-gliadins , LMW-glutenins , HMW-secalins , γ-40 secalins and/or γ-75 secalins. The gluten proteins other than the at least one peptide or protein may alternatively or additionally include glutelins.

The composition may further comprise at least one addi ^¬ tional inert protein. The term "inert protein" may, in the con ^¬ text of this specification, refer to any protein that does not significantly bind to an antibody used for quantifying gluten, such as the antibody R5, and/or that is not otherwise detectable in a gluten assay, such as an ELISA assay. The inert protein may also be essentially soluble in an alcohol containing solution. An example of such an inert protein may be e.g. BSA (bovine se- rum albumin) ; however, a skilled person is aware of various other suitable inert proteins and is also capable of selecting oth ^¬ er suitable inert proteins. The inert protein may be a non- gluten protein. The inert protein may be included in the compo ^¬ sition so that the composition mimics total gluten or total hor- dein more closely.

The composition may comprise at least 5 w-%, or at least 10 w-%, or at least 20 w-%, or at least 30 w-%, or at least 40 w-%, or at least 20 w-%, or 5 to 50 w-%, or 10 to 30 w- %, or 5 to 20 w-% of the at least one peptide or protein by weight of the total dry peptide and protein in the composition. In an embodiment, the composition may comprise about 5 to 15 w-% of the at least one peptide or protein by weight of the total dry peptide and protein in the composition. In an embodiment, the composition may comprise about 25 to 35 w-% of the at least one peptide or protein by weight of the total dry peptide and protein in the composition. In embodiments in which the at least one peptide or protein consists of one or more proteins, the "total dry peptide and protein" may be understood as referring to the total dry protein in the composition. The relative amount of the at least one peptide or protein may be selected e.g. on the basis of the assay in which the composition is used as a reference material. For instance, for a sandwich or competitive ELISA assay, a different relative amount of the at least one peptide or protein may be selected. For a sandwich ELISA assay using R5 as the antibody, the composition may comprise, for ex ^¬ ample, about 25 to 35 w-% of the at least one peptide or protein by weight of the total dry protein in the composition. For a competitive ELISA assay using R5 as the antibody, the composi ^¬ tion may comprise, for example, about 5 to 15 w-% of the at least one peptide or protein by weight of the total dry protein in the composition.

The relative amount of the at least one purified pep ^¬ tide or protein may also be selected on the basis whether the source of the gluten contamination is barley, wheat or rye.

For example, in an embodiment, the composition may com ^¬ prise about 30 to 50 w-% of the at least one peptide or protein, for example C-hordein, by weight of the total dry peptide and protein in the composition. Such a composition may be suitable for quantifying the amount of gluten present in a food sample, wherein the source of the gluten in the food sample comprises or is barley. In an embodiment of the use of the composition as a reference material for quantifying the amount of gluten present in a food sample or of the method for quantifying the amount of gluten present in a food sample, the composition comprises about 30 to 50 w-% of the at least one peptide or protein, for example C-hordein, by weight of the total dry peptide and protein in the composition, and the source of the gluten in the food sample comprises or is barley. About 40 w-% of C-hordein may be consid- ered to represent an average for barley cultivars relatively well .

In an embodiment, the composition may comprise about 10 to 35 w-% of the at least one peptide or protein, for example C- hordein, by weight of the total dry peptide and protein in the composition. Such a composition may be suitable for quantifying the amount of gluten present in a food sample, wherein the source of the gluten in the food sample comprises or is wheat. In an embodiment of the use of the composition as a reference material for quantifying the amount of gluten present in a food sample or of the method for quantifying the amount of gluten present in a food sample, the composition comprises about 10 to 35 w-% of the at least one peptide or protein, for example C- hordein, by weight of the total dry peptide and protein in the composition, and the source of the gluten in the food sample comprises or is wheat.

In an embodiment, the composition may comprise about 10 to 35 w-% of the at least one peptide or protein, for example C- hordein, by weight of the total dry peptide and protein in the composition. Such a composition may be suitable for quantifying the amount of gluten present in a food sample, wherein the source of the gluten in the food sample comprises or is rye. In an embodiment of the use of the composition as a reference mate ^¬ rial for quantifying the amount of gluten present in a food sam ^¬ ple or of the method for quantifying the amount of gluten pre- sent in a food sample, the composition comprises about 10 to 35 w-% of the at least one peptide or protein, for example C- hordein, by weight of the total dry peptide and protein in the composition, and the source of the gluten in the food sample comprises or is rye.

In the above embodiments, the remaining portion of the total dry peptide and protein in the composition may comprise or consists of an inert protein, for example BSA. Such compositions may more accurately reflect the gluten compositions of barley, wheat or rye. A kit comprising the composition according to one or more embodiments described in this specification and option ^¬ ally instructions for use is also disclosed. The kit and/or the instructions for use may be for quantifying the amount of gluten present in a food sample, for instance according to the method according to one or more embodiments described in this specifi ^¬ cation. The kit and/or the instructions for use may also be for improving the accuracy of quantifying the amount of gluten pre- sent in the food sample.

Use of at least one purified peptide or protein accord ^¬ ing to one or more embodiments described in this specification or of the composition according to one or more embodiments de ^¬ scribed in this specification as a reference material for quan- tifying the amount of gluten present in a food sample is also disclosed. In an embodiment, the use may be use for improving the accuracy of quantifying the amount of gluten present in the food sample. The food sample may be obtainable from a food that is assumed gluten-free but contaminated with gluten; from a food that contains gluten; from a food that contains residual gluten; and/or from a food that is gluten-free and not contaminated with gluten. Thus the purified peptide or protein or the composition may be used for quantifying residual and/or contaminating gluten. The purified peptide or protein or the composition may be used as an internal and/or external standard.

A method for purifying at least one peptide or protein from a starting material is also disclosed, comprising

extracting at least a portion of the at least one pep ^¬ tide or protein present in the starting material with an aqueous solution comprising an alcohol; and

purifying the at least one peptide or protein from the aqueous solution to obtain a composition comprising the at least one peptide or protein, wherein the at least one peptide or pro ^¬ tein comprises at least one amino acid stretch having the se- quence QQPFP (SEQ ID NO: 1) .

The method may optionally comprise removing at least a portion of albumin and/or globulin proteins present in the starting material prior to extracting at least a portion of the at least one peptide or protein. The at least a portion of the albumin and/or globulin proteins may be removed using the Os ^¬ borne extraction sequence. An example of a suitable Osborne ex- traction sequence is described in Example 1. Other methods may also be contemplated.

The alcohol may be, for example, ethanol, propanol or any mixtures or combinations thereof, but other alcohols may al ^¬ so be contemplated. The aqueous solution may comprise about 20 to 70 v-% of the alcohol. In an embodiment, the aqueous solution comprises about 30 to 60 v-% of the alcohol.

The at least one peptide or protein may comprise a plu ^¬ rality of amino acid stretches having the sequence QQPFP (SEQ ID NO: 1) .

The at least one peptide or protein may comprise at least one amino acid stretch having the sequence PQQPFPQQ (SEQ ID NO: 2) .

The at least one peptide or protein may comprise a plu ^¬ rality of amino acid stretches having the sequence PQQPFPQQ (SEQ ID NO: 2) .

The at least one peptide or protein may comprise at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13 amino acid stretches having the sequence QQPFP (SEQ ID NO: 1) .

The at least one peptide or protein may comprise at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13 amino acid stretches having the sequence PQQPFPQQ (SEQ ID NO: 2) .

In an embodiment, the at least one peptide or protein is sulphur poor.

The at least one peptide or protein may comprise or be at least one prolamin protein. The at least one peptide or pro ^¬ tein may be at least one native prolamin protein. Native prola ^¬ min proteins are obtainable from barley, wheat or rye grains us- ing a suitable method, such as the method for purifying at least one peptide or protein according to one or more embodiments de ^¬ scribed in this specification. In an embodiment, the at least one prolamin protein is sulphur poor. The at least one peptide or protein may comprise or be at least one prolamin protein selected from the group consisting of C-hordein, ω-gliadin, ω-secalin and any combinations or mix- tures thereof.

In an embodiment, the at least peptide or protein com ^¬ prises or is C-hordein. C-hordein is relatively readily obtaina ^¬ ble from barley grains .

S-poor prolamins, such as C-hordein of barley, ω- gliadin of wheat and ω-secalin of rye, typically consist essen ^¬ tially or mainly of the repeats, i.e. amino acid stretches, PQQPFPQQ (SEQ ID NO: 2) and non-repetitive sequences at the isl ^¬ and C-termini.

However, the presence of non-repetitive sequences with- in the at least one peptide or protein and/or at the N- and C- terminals of the at least one peptide or protein are not exclud ^¬ ed .

In an embodiment, the aqueous solution with which the at least a portion of the at least one peptide or protein pre- sent in the starting material is extracted does not comprise at least one of a reducing agent or a dissociating agent.

In an embodiment, the aqueous solution with which the at least a portion of the at least one peptide or protein pre ^¬ sent in the starting material is extracted does not comprise a reducing agent. In the context of this specification, the term "reducing agent" may be a disulfide reducing agent. The disul ^¬ fide reducing agent may be any agent capable of reducing disul ^¬ fide groups. Examples of such reducing agents may be e.g. 2- mercaptoethanol , dithiothreitol , tris (2-carboxyethyl) -phosphine, sodium borohydride and any mixtures or combinations thereof. However, other reducing agents may also be contemplated, for ex ^¬ ample other borohydrides , diborane, iron (II) compounds, tin (II) compounds, dithionates, thiosulfates , H ₂ 0 ₂ , hydrazines, oxalic acid, formic acid, ascorbic acid, reducing sugars, hypophos- phites, and any mixtures or combinations thereof.

In an embodiment, the aqueous solution with which the at least a portion of the at least one peptide or protein pre ^¬ sent in the starting material is extracted does not comprise a dissociating agent. In the context of this specification, the term "dissociating agent" may refer to any agent that may disso ^¬ ciate proteins by disrupting protein conformation, i.e. break down protein quaternary, tertiary and/or secondary structures. Examples of dissociating agents may be urea, guanidine hydro ^¬ chloride, sodium dodecyl sulphate, N-lauroylsarcosine and other detergents, and any mixtures or combinations thereof. In an em ^¬ bodiment, the dissociating agent is urea. Urea and other disso- dating agents included in the aqueous buffer may disrupt the conformation of the peptide or protein in an undesirable manner. They may also interfere with the determination of the amount of the extracted protein.

In an embodiment, the aqueous solution with which the at least a portion of the at least one peptide or protein pre ^¬ sent in the starting material is extracted does not comprise ei ^¬ ther of a reducing agent and a dissociating agent. In other words, it does not comprise a reducing agent and it does not comprise a dissociating agent.

The at least one peptide or protein may be purified from the aqueous solution using ion exchange chromatography, reverse phase chromatography, size exclusion chromatography or any combinations thereof.

The starting material may be a cereal material. The ce- real material may be material from the Triticeae cereals, such as flour, obtainable from grains of Triticeae species. The mate ^¬ rial may thus be obtainable from wheat, rye, or barley, or any combinations thereof. The cereal material may be comminuted, for instance ground or milled. The starting material may, in other embodiments, be any other suitable starting material, such as a host cell or a growth medium used for the production of the at least one peptide or protein.

In an embodiment, the starting material is a Triticeae material, such as flour, obtainable from a hulless cultivar. The hulless cultivar may be a hulless barley cultivar. An example of such a hulless barley cultivar is cv.'Jorma', but other hulless Triticeae cultivars are also available. The hulless cultivar may obviate the need to remove the hull, thus simplifying the ex- traction. The term "hulless" may refer to a ^x naked' cultivar in which the hull is more loosely held onto the seed than in a cov ^¬ ered cultivar, such that the hull typically falls off during harvesting.

In an embodiment, the composition comprises at most 5 w-%, or at most 3 w-%, or at most 2 w-%, or at most 1 w-% of gluten proteins other than the at least one peptide or protein by weight of the total dry peptide and protein in the composi- tion.

In an embodiment, the composition may comprise about 30 to 50 w-% of the at least one peptide or protein, for example C- hordein, by weight of the total dry peptide and protein in the composition. Such a composition may be suitable for quantifying the amount of gluten present in a food sample, wherein the source of the gluten in the food sample comprises or is barley. In an embodiment, the composition may comprise about 10 to 35 w- % of the at least one peptide or protein, for example C-hordein, by weight of the total dry peptide and protein in the composi- tion. Such a composition may be suitable for quantifying the amount of gluten present in a food sample, wherein the source of the gluten in the food sample comprises or is wheat. In an em ^¬ bodiment, the composition may comprise about 10 to 35 w-% of the at least one peptide or protein, for example C-hordein, by weight of the total dry peptide and protein in the composition. Such a composition may be suitable for quantifying the amount of gluten present in a food sample, wherein the source of the glu ^¬ ten in the food sample comprises or is rye. In the above embodi ^¬ ments, the remaining portion of the total dry peptide and pro- tein in the composition may comprise or consists of an inert protein, for example BSA. The method may thus further comprise adjusting the amount of the at least one peptide or protein to the desired amount.

A method for quantifying the amount of gluten present in a food sample is disclosed, the method comprising extracting at least a portion of the gluten from the food sample, contact ^¬ ing the extracted gluten with an antibody, thereby detecting the amount of the gluten; wherein the method comprises quantifying the amount of gluten in the food sample using the composition according to one or more embodiments described in this specifi ^¬ cation or the at least one purified peptide or protein according to one or more embodiments described in this specification as a reference material.

The food sample may be obtainable from a food that is assumed gluten-free but contaminated with gluten; from a food that contains gluten; from a food that contains residual gluten; and/or from a food that is gluten-free and not contaminated with gluten. Thus the method may be suitable for quantifying the amount of residual and/or contaminating gluten.

The antibody may be R5. However, other antibodies may also be contemplated.

The detecting the amount of the gluten by the antibody may be performed by an ELISA assay. The ELISA assay may be, for example, a sandwich ELISA assay or a competitive ELISA assay. However, other ELISA assays may also be contemplated. In the ELISA assay, the antibody may be R5.

The reference material, i.e. the at least one peptide or protein or the composition, may be used as an external and/or as an internal standard. An absolute or a relative amount of the gluten may be quantified.

The method may comprise spiking the food sample or the extracted gluten with the composition according to one or more embodiments described in this specification or with the at least one purified peptide or protein according to one or more embodi ^¬ ments described in this specification.

The method may be a method for quantifying the amount of residual gluten or gluten present as a contamination in the food sample, wherein the relative amount of the at least one pu ^¬ rified peptide or protein is selected based on the source of the contamination and/or the ELISA assay. The relative amount of the at least one purified peptide or protein may be selected as de- scribed above. The composition may comprise at least 5 w-%, or at least 10 w-%, or at least 20 w-%, or at least 30 w-%, or at least 40 w-%, or at least 20 w-%, or about 5 to 50 w-%, or about 10 to 30 w-%, or about 5 to 20 w-%, or about 5 to 15 w-%, or about 25 to 35 w-% of the at least one peptide or protein by weight of the total dry peptide and protein in the composition. The relative amount of the at least one purified peptide or pro- tein may also be selected on the basis whether the source of the gluten contamination is barley, wheat or rye. The relative amount of the at least one peptide or protein may be selected e.g. on the basis of the assay in which the composition is used as a reference material. For instance, for a sandwich or compet- itive ELISA assay, a different relative amount of the at least one peptide or protein may be selected. For a sandwich ELISA as ^¬ say using R5 as the antibody, the composition may comprise, for example, about 25 to 35 w-% of the at least one peptide or pro ^¬ tein by weight of the total dry peptide and protein in the com- position. For a competitive ELISA assay using R5 as the anti ^¬ body, the composition may comprise, for example, about 5 to 15 w-% of the at least one peptide or protein by weight of the to ^¬ tal dry protein in the composition.

C-hordein, the primary structure of which is mainly formed of repeating amino acid stretches of PQQPFPQQ (SEQ ID NO: 2), and other peptides or proteins comprising a plurality of the repeats QQPFP (SEQ ID NO: 1) and/or PQQPFPQQ (SEQ ID NO: 2) are strongly recognized by the R5 antibody. C-hordein may thus be 15-20 times more reactive than the currently commonly used ref- erence gliadin. Therefore peptides or proteins comprising one or more of the amino acid streches, i.e. repeats, may be a rela ^¬ tively uniform, sensitive and practical reference material for the detection and quantification of gluten, in particular of residual gluten or gluten present in a food or food sample due to contamination. The peptide or protein is also relatively soluble and thus may be kept in a soluble state for storage and/or anal ^¬ ysis. Being more homogeneous than e.g. whole hordein or various other prolamin and/or gluten fractions, it may be used for calibrating the gluten, in particular the hordein, concentration in products, such as oats and oat products, containing barley.

It will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments. The embodiments are not limited to those that solve any or all of the stated problems or those that have any or all of the stated benefits and advantages. It will fur ^¬ ther be understood that reference to 'an' item refers to one or more of those items.

The term "comprising" is used in this specification to mean including the feature (s) or act(s) followed thereafter, without excluding the presence of one or more additional fea ^¬ tures or acts.

EXAMPLES

The description below discloses some embodiments of the invention in such a detail that a person skilled in the art is able to utilize the invention based on the disclosure. Not all steps of the embodiments are discussed in detail, as many of the steps will be obvious for the person skilled in the art based on this specification.

EXAMPLE 1 - Preparing hordein fractions for reference material

Materials

Twenty-nine barley cultivars for feeding and malting purposes were selected for this study. ^x Brage' , ^x Tocada' , ^x Vilde' , ^x Marthe' , ^x Fairytale' , ^x Aukusti' , ^x Wolmari' , ^x Barke' , ^x Polartop', ^x Einar' , Vyva' , ^x Saana' , ^x Edvin' , ^X NFC Tipple', ^x Streif' , ^x Harbinger' , ^x Scarlett' and ^x Elmeri' were kindly pro ^¬ vided by Boreal Plant Breeding Ltd. (Finland) . ^x Xanadu' by Kesko Corporation (Finland) , and ^x Propino' by Syngenta (Finland) . These were the top 20 cultivars in Finland during 2012-2015. In addition, ^X KWS Asta' , ^x Lacey' , ^x Minttu' , ^x Toria' , 'Overture', ^x Voitto', 'Annabel' and ^X SW Mitja' (Boreal Plant Breeding Ltd.) were analysed. Hulless cv. Vorma' was from Risto Laitinen (Vil- lala, Finland) . The barley kernels were milled to flours with a Brabender Quadrumat Junior (Duisburg, Germany) and passed through an 850 μιη sieve.

All the chemicals used were analytical quality or bet ^¬ ter. Bovine serum albumin (BSA) with purity ≥ 98% was purchased from Santa Cruz Biotechnology (Dallas, Texas) . Hordein composition by reserve-phase high-pressure liquid chromatography (RP-HPLC)

Hordein composition of selected barley flour (0.1 g) was extracted following the Osborne sequence 1 ml of mQ-water, 1 ml of 0.5 M NaCl, and 1 ml of mQ-water to remove the albumin and globulin proteins at ambient temperature. After each extraction, samples were centrifugated for 10 min at 20000 xg was applied. The pellet was extracted either once or 3 times with 40% (v/v) 1-propanol with 5% (v/v) 2-mercaptoethanol at 50°C for 30 min to provide the whole hordein extract. The barley flour was also di ^¬ rectly extracted 1 time or 3 times with the same solvent. After centrifugation, the supernatant was filtered through a 0.45 μιη membrane and was ready for RP-HPLC separation of the hordein fractions. An injection volume of 10 μΐ of whole hordein was separated on a C8 column (Dimension, company, details) , where the temperature was kept at 35°C. An acetonitrile gradient from 20% solvent B (0.1% TFA in acetonitrile) to 50% solvent A (0.1% TFA in milli-Q water) was run over 40 min at a flow rate of 0.7 ml/min, and the elution was monitored at 210 nm. The hordein fraction peaks were integrated and the peak areas were compared to determine the C-hordein proportion of total hordein in each cultivar. Because the proportion of γ-hordein was reported to be very minor, and the HPLC profile did not distinguish it, it was counted together with B-hordein proportion.

Hordein HPLC profile and C-hordein proportion

When whole hordein was separated on a C8 column (D- hordein was first eluted, followed by C-hordein and B-hordein (Figure 1, Marchylo & Kruger 1984) . According to the peak pat ^¬ tern of C-hordein, the 29 analysed cultivars can be divided into three patterns, and of which type (b) , for example cv. Barke, was the most common present in 19 cultivars. The C-hordein pro ^¬ portion in total hordein of cv. Elmeri (2015) after a single ex- traction was 26.0% and after three sequenced extracts was 24.7%; the corresponding figures after Osborne extraction were 25.5% and 25.7%. There was no significant difference between the sin ^¬ gle and triple extraction results, the C-hordein proportion of all cultivars was determined with a single direct extraction (Table 1) .

Figure 1 shows a reverse-phase liquid chromatogram of total hordein extract separated on a C8 column, (a) cv. Harbin ^¬ ger; (b) cv. Barke; (c) cv. NFC Tipple. The total hordein was directly extracted with 40% (v/v) 1-propanol with 5% (v/v) 2- mercaptoethanol from barley flour.

From 2012 to 2015, ^x Barke' and ^X NFC Tipple' were the two most popular malting cultivars in Finland, covering over 50% of the harvested area of this crop. Feeding cultivars were far more diverse, with 'Elmeri' , ^x Wolmari' , ^x Brage' and ^x Aukusti' took over over 40% (National Land Survey of Finland, 2012- 2015) . The C-hordein proportion of the 29 cultivars ranged two fold, from 16.5% to 33.1% (Table 1) . There was slight variation in the C-hordein proportion of cv. Elmeri from 2010, 2014 and 2015. From this data set, the weighted average C-hordein propor ^¬ tion of the whole hordein in Finland 2012-2015 was 25-26%. Table 1 C-hordein content as percentage of whole hordein of se ^¬ lected barley cultivars.

Culti- Year Origin ConCulti- Year Origin Convar tent var tent

(%) (%)

KWS 2013 K-Eur 16.5 Saana 2014 Scand 27.5

Asta

Marthe 2009 17.4 Edvin 2010 Scand 28.0

Tocada 2013 K-Eur 18.6 Elmeri 2015 Scand 28.1

Vilde 2010 Scand+E 18.6 NFC 2013 K-Eur 28.1

ur Tipple

Pro- 2014 20.3 Brage 2010 Scand 28.3

pino

Fair2013 K-Eur 20.7 Streif 2014 28.5

ytale

Toria 2010 Scand 22.6 Minttu Scand 28.8

Over2013 K-Eur 23.0 Elmeri 2014 Scand 29.2

ture

Aukus- 2010 Scand 23.1 Har- 2014 Scand 29.6 ti binger

Wol- 2010 Scand 23. 3 Scar2013 K-Eur 30. 0

mari lett

Barke 2013 K-Eur 24. 5 Jorma Scand 30. 5

Po- 2010 24. 5 Voitto 2010 Scand 31. 1

lartop

Einar 2010 Scand 25. 6 Xanadu 2014 31. 1

Jyva Scand 25. 6 Lacey 2010 NorAm 32. 8

SW 2014 Scand 25. 7 Elmeri 2010 Scand 33. 1

Mitja

Anna ^¬ 20 25. 9

bel

Hordein fractions reactivity against R5 antibody

The hordein fractions were collected, and their protein content was quantified with a BSA standard (linear range 0-60 μg) by peak area. The collected fractions were dried first under a nitrogen flow and then under vacuum centrifugation (Savant SpeedVac SCllOA concentrator, USA) . The dried fractions were solubilized in cocktail solution (R7006, R-Biopharm, Darmstadt, Germany) and analyzed with R5 sandwich ELISA (R7001, R-Biopharm, Darmstadt, Germany) .

Hordein subunits reactivity against R5 antibody

Figure 2 illustrates the reaction of isolated hordeins against R5 antibody in sandwich ELISA. Three types of C-hordein and B-hordein were from cultivars ^x Harbinger' , ^X NFC Tipple' and ^x Barke' . D-hordein was from ^x Harbinger' .

The reactivity of D-, C- and B-hordeins against R5 an ^¬ tibody varied widely in sandwich ELISA (Figure 2) . Because the amount of antibody is fixed in the test kit, the less lower the concentration of protein required to achieve a certain absorb- ance, the more sensitive the reaction. By this measure, C- hordein was 10-20 times more reactive than gliadin standard, which in turn was 8-25 times more reactive than B-hordein. The slope of the curve indicated that C-hordein and gliadin standard had similar affinity with R5 antibody, while B-hordein had less, and D-hordein had almost none. The three types of C-hordein and B-hordein reacted similarly with R5 antibody, although their HPLC patterns were different.

Purification of C-hordein in a preparative scale

The flour of cv.'Jorma' was first washed with acetone and filtered, then extracted following the Osborne sequence de- scribed earlier. The monomeric hordein was extracted with 40% (v/v) 1-propanol at 50°C without reducing agent. After centrifu- gation at 18000 xg, the supernatant was left at 4°C for over ^¬ night. The suspension was centrifuged again to remove the pre ^¬ cipitates before mixing with buffer A. The buffer A consisted of 40% (v/v) 1-propanol, 10 mM glycine, and 50 mM citric acid- sodium citrate buffer at pH 3.0, and buffer B consisted of 40% (v/v) 1-propanol, 0.5 M NaCl, 10 mM glycine, and 50 mM citric acid-sodium citrate buffer at pH 3.0. An ion exchange column (35 mm diameter ^χ 300 mm length) was packed with TOYOPEARL SP-650M (TOSOH Bioscience LLC, Japan) and coupled with AKTAprime plus system (GE Healthcare, Sweden) . After the sample was loaded, the separation gradient was 0-50% B from 0 ml to 600 ml at a flow rate of 15 ml/min. The elution was collected as 10 ml per frac ^¬ tion, and monitored at 280 nm. The components of fractions were analyzed by SDS-PAGE (NuPage Bis-Tris 10%, Invitrogen, LifeTech- nologies) . The fractions containing C-hordein were combined and dialyzed against mQ-water with cut-off MW 10 000 ( Sigma-Aldrich D9527). The dialysed C-hordein was lyophilized (Heto-Holton DW8- 85, Denmark) . For the purpose of this study, this fraction was used as a benchmark of ^x pure' C-hordein.

Preparation of C-hordein by FPLC

Figure 3 (a) shows a cation-exchange chromatogram of hordein. The blue line shows the absorbance at 280 nm, with fractions 13, 19 and 54 were circled, and the red line shows the conductivity. The inset shows SDS-PAGE of collected fractions, fraction; (b) Reverse-phase chromatogram of purified C-hordein and whole hordein from cv. Vorma ' . When hordein was extracted with 40% 1-propanol without reducing agents and kept in 4°C overnight, precipitates appeared that were mainly composed of B-hordein. At pH 3, C-hordein and B-hordein were clearly separated in cation-exchange chromatog ^¬ raphy (Figure 3a) . Fractions 11-15 absorbed at 280 nm but did not contain prolamins, indicating that they were other compounds. Fractions 17-25 contained C-hordein and were collected and combined in native form. Following increased ion exchange, B-hordein started to elute at fractions 54-57 (Figure 3a) . HPLC of lyophilized C-hordein shows its purity in comparison with the whole hordein (Figure 3b) .

EXAMPLE 2 - Purified C-hordein as reference material in ELISA

C-hordein as a reference material in sandwich and competitive ELISA

Lyophilized C-hordein and BSA were dissolved separately in 60% (v/v) ethanol at 0.3 mg/ml, and then mixed in ratios of 1:9 (C-hordein : BSA) , 2:8, 3:7, 4:6 and 5:5, further referred as 10%, 20%, 30%, 40%, 50% C-hordein at a constant protein concen ^¬ tration of 0.3 mg/ml. Because BSA does not react with R5 anti ^¬ body, C-hordein mixed with BSA in different ratios to mimic to ^¬ tal hordein in ELISA. These samples were tested in sandwich and competitive ELISA (R7021, R-Biopharm, Darmstadt, Germany) to compare with the kit reference materials.

Figure 4 illustrates the reaction of C-hordein mixed with bovine serum albumin (BSA) in different ratios against R5 antibody (a) in sandwich ELISA; and (b) in competitive ELISA. (c) Reaction of purified whole hordein of 6 cultivars in R5 sandwich ELISA compared with 30%, 40% and 50% C-hordein stand ^¬ ards and gliadin standard.

In sandwich ELISA, the affinity (the slope) of C- hordein with R5 antibody behaved similarly with gliadin stand- ard, and at a ratio of 3 C-hordein: 7 BSA (30% C-hordein), the reaction almost matched that of the gliadin standard. In compet ^¬ itive ELISA, the affinity of C-hordein with R5 antibody also was similar to that of the peptide standard, and at a ratio of 1 C- hordein: 9 BSA (10% C-hordein) was closest to the peptide stand ^¬ ard. Using the C-hordein and BSA in different ratios could re ^¬ place both the gliadin standard in R5 sandwich ELISA, and the peptide standard in R5 competitive ELISA. In sandwich ELISA, the curves of purified whole hordein of common cultivars, such as ^x Barke' and ^X NFC tipple' (C-hordein proportions 24.5% and 28.1% respectively) , were above that of the gliadin standard and be ^¬ tween that of the 30% and 50% C-hordein standard. The curve of ^X KWS Asta' , with its low C-hordein proportion (16.5%), was close to the gliadin standard curve and that of 30% C-hordein.

EXAMPLE 3 - Measuring barley contamination in oat Prolamin quantification of oat flour spiked with barley flour

Barley flour of cultivars ^x Elmeri' , ^x Einar' and ^x Marthe' with different C-hordein proportions (33.1%, 25.6%, 17.4%) were selected for spiking in gluten-free oat flour. An aliquot of 0.1 g of barley flour added to 0.4 g whole grain oat flour (Provena, Raisio Nutrition Ltd. Finland) and homogenized by vortexing. The mix was further diluted to 400 times by oat flour manually in steps ( ^χ 4, ^χ ΐθ, ^χ ΐθ) to ensure homogeneity. The hordein concentration was also determined by R5 sandwich ELISA, and calculated with gliadin reference material and 40% C- hordein. The spiking was conducted in three replicates and each replicate was measured in two dilutions, and the means and standard errors were calculated. The prolamin concentration of the three barley cultivars was also determined by RP-HPLC using BSA as standard. Flour was extracted by 40% (v/v) 1-propanol with 5% (v/v) 2-mercaptoethanol and injected in a C8 column as described earlier.

Figure 5 shows the prolamin concentration of gluten- free oat flour spiked with three barley flours, determined by HPLC, R5 sandwich ELISA with 40% C-hordein standard, and R5 sandwich ELISA with gliadin standard.

With a constant spiking amount, the determined prolamin concentrations of three barley flours were different in HPLC, R5 sandwich with gliadin standard, and R5 sandwich with 40% C- hordein standard, but in the same order of ^x Elmeri' >' Einar' >' Marthe' . The prolamin concentration calibrated by gliadin standard was 2.5 times ( ^x Elmeri' ) , 1.8 times ( ^x Einar' ) , and 1.2 times ( ^x Marthe' ) the HPLC results. However, calibrated by 40% C-hordein standard, the estimated prolamin concentration was 1.2 times ( ^x Elmeri' ) , 0.85 times ( ^x Einar' ) and 0.63 times ( ^x Marthe' ) the HPLC results. For ^x Elmeri ^λ and ^x Einar' , the estimated value by the 40% C-hordein standard were not significantly different from those determined by HPLC, but for ^x Marthe' the estimate was significantly lower, until the standard was changed to 30% C-hordein.

The above examples investigated the reason for the overestimation of barley prolamin when measured by R5 antibody ELISA with the gliadin standard. C-hordein was 80-200 times more reactive with R5 antibody than B-hordein, whereas D-hordein did not react significantly with R5 antibody. When detecting whole hordein with R5, the main recognition was from C-hordein, so the proportion of C-hordein in whole hordein was crucial for correct prolamin quantification. The C-hordein proportion varied from 16.5% to 33.1% in the common barley cultivars in Finland, with an average C-hordein around 26%. The composition of the gliadin standard was not comparable to the composition of hordein. Using the gliadin standard caused a 1.8-2.5 times overestimation of barley flour spiked in gluten-free oat flour, but use of a 40% C-hordein standard allowed calibration to give the correct con ^¬ centration .

The varying reactivity of hordein subunits against R5 antibody is at least partially attributable to the number of epitopes. The main R5 epitope, QQPFP (SEQ ID NO: 1), appeared 13 times in C-hordein (Uniprot Q40055, SEQ ID NO: 3), and minor epitopes QQPYP (SEQ ID NO: 4), QQTFP (SEQ ID NO: 5), PQPFP (SEQ ID NO: 6) and QLPFP (SEQ ID NO: 7) appeared once each. One main epitope QQPFP (SEQ ID NO: 1) and 7 minor epitopes were found in B3 hordein (Uniprot I6TEV5, SEQ ID NO: 8), and 5 QQPFP epitopes in Bl hordein (Uniprot P06470, SEQ ID NO: 9) . Only one QQPFP epitope was found in X3-hordein (Uniprot P80198, SEQ ID NO: 10) and no R5 epitope was found in D-hordein (Uniprot Q84LE9, SEQ ID NO: 11) .

The present results showed that the C-hordein of the common European cultivars ^x Harbinger' , ^X NFC Tipple' and ^x Barke' , behaved similarly in the recognition of the R5 antibody, alt ^¬ hough their HPLC profile differed. C-hordein of a single culti- var can consist of up to 20 polypeptides, thus forming different HPLC profiles. The primary structures of these prolamin polypep- tides are highly homologous, but their molecular weights differ (Tatham, Drake & Shewry, 1989) . In this study, three groups of C-hordein were gathered according to their RP-HPLC profiles.

The ratio of B-hordein to C-hordein according to the present results ranged from 1.89 of cv. ^x Elmeri' to 4.68 of cv. ^X KWS Asta' . The corresponding protein group of C-hordein in wheat is ω-gliadin, which shows about 70% homology with it, and its repetitive sequence in the central domain is PFPQQPQQ (SEQ ID NO: 12) . The ω-gliadin proportion of total gliadin has been reported to range from 6.2% to 20.0% (Wieser, Seilmeier & Be- litz, 1994), and from 10% to 19% (Daniel & Triboi, 2000). The proportion of C-hordein in total hordein is generally higher than the proportion of ω-gliadin in total gliadin. The composition of prolamin may be affected by cultivar, environment and nitrogen nutrition.

A C-hordein standard could be applied to determine the prolamin content in the circumstance like (1) contamination in oats; (2) barley starch, (3) malt and malt extract. Contamina ^¬ tion of oat may come from wheat, barley, rye, or their mixture. The resource of contamination may be relevant in order to achieve correct quantification, by other methods like PCR. If the contaminant is wheat, either a gliadin standard or 30% C- hordein can be applied for calibration. If the contaminant is barley, which is more likely, a standard comprising e.g. C- hordein, based on the average C-hordein proportion of the re- gion, can be used for calibration. In the case of measuring prolamin content of barley starch, the process is under low pH so that acid hydrolysis of hordein may occur, so competitive ELISA may be utilized to detect small fragments, along with the sand- wich assay. Barley malt, which is partially hydrolyzed by its endogenous enzymes, may be tested in both sandwich and competi ^¬ tive assays. In the sandwich assay, the current commercial ref- erence material is the gliadin standard, while in the competi ^¬ tive assay it is a hydrolysate mixture of the gliadin standard, one barley cultivar and one rye cultivar (Gessendorfer, Koehler & Wieser, 2009) . Clearly, these two reference materials are not comparable. In addition, the commercial peptide standard was hy- drolyzed by the digestive enzymes pepsin and trypsin, which is very different from the malting or the starch separation process. The present results showed that an exemplary 30% C-hordein and BSA mixture could represent gliadin standard, while the 10% C-hordein mixture can represent the peptide standard. Thus, the use of the standard could not only allow correct determination of prolamin content, but also make the results from sandwich and competitive assays comparable.

For gluten analysis, the prolamin working group (PWG) gliadin standard is available, however it is not reproducible, and it is not accepted as certified reference material in the Institute of Reference Material and Measurements of the European Commission due to its high glutenin content (Working Group on Prolamin Analysis and Toxicity, 2016).

In conclusion, the present results have determined that the high proportion of C-hordein in total hordein may result for the consistent overestimation of hordein by the R5 ELISA which uses gliadin as reference material in gluten-free analysis.

It is obvious to a person skilled in the art that with the advancement of technology, the basic idea of the invention may be implemented in various ways. The invention and its embod ^¬ iments are thus not limited to the examples described above; in ^¬ stead they may vary within the scope of the claims.