Cereals for developing gluten-free products and analytical tools for gluten detection

CEREALS FOR DEVELOPING GLUTEN-FREE PRODUCTS AND ANALYTICAL TOOLS FOR GLUTEN DETECTION Cristina M. Rosell1*, Francisco Barro2, Carolina Sousa3, Mª Carmen Mena4 1Institute of Agrochemistry and Food Technology. CSIC. Avenida Agustin Escardino, 7. Paterna 46980. Valencia. Spain. 2 Institute for Sustainable Agriculture. CSIC. Alameda del Obispo s/n, 14080-Córdoba, Spain. 3 Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain. 4 Proteomics Facility, Centro Nacional de Biotecnología. CSIC. Calle Darwin 3, Cantoblanco, 28049 Madrid, Spain. Address for correspondence: *[email protected] Abstract Recently, gluten free foods have attracted much research interest motivated by the increasing market. Despite the motivation for developing gluten-free foods it is necessary to have a scientific basis for developing gluten-free foods and the tools for detecting the peptide sequence that could be immune-toxic to some persons. This review will be focused primarily on the cereal-based commodities available for developing gluten free blends, considering those naturally gluten free cereals besides the controversial oats, and the recent transgenic approaches for developing cereals free of immunotoxic gluten. Secondly, the biochemical tools for mimicking gluten network viscoelastic properties will be presented. Finally, special emphasis will be put in compiling the available techniques for gluten detection and quantitation. Key words: gluten free cereals, breeding, analytical tools, prolamins 1. Introduction Wheat, barley and rye are temperate cultivated cereals from the grass tribe triticeae. These cereals are major suppliers of calories to the human diet as the main component of the grain is starch. They also contain 8-15% of protein, which mainly correspond to the gluten proteins. Those proteins are important because of their impact on grain processing properties, for the production of bread, pasta and noodles from wheat, and for malting, brewing and distilling of barley. Gluten is a complex mixture of proteins comprising the gliadins and glutenins in wheat and equivalent proteins in barley and rye, representing 80% of total grain proteins. However, gluten proteins also have negative impacts on human health, in relation to allergies and intolerances. Gluten is defined differently depending on the discipline (biochemistry, food technology, nutrition). In the gluten-free intolerance context the most appropriate definition is that provided by the European Commission where “gluten means a protein fraction from wheat, rye, barley, oats or their crossbred varieties and derivatives thereof, to which some persons are intolerant and which is insoluble in water and 0.5 M sodium chloride solution” (Commission of the European Communities, 2009). In recent decades, gluten has attracted great attention due to the increasing number of diagnosed patients with intolerance to this protein fraction, relating to the improved sensitivity of the detection methods and the increasing awareness of the existence of the disease. Three pathologies are associated with gluten intake (Figure 1), which appear to be increasing in importance: i) food allergy that affects 0.2-0.5% of the population but has stronger clinical implications (Zuidmeer et al., 2008), ii) coeliac disease (CD), which is an autoimmune disorder caused by the ingestion of gluten not only from wheat, but also rye, barley and some varieties of oats (Comino et al., 2011) affects both children and adults throughout the world at various frequencies (from 0.1% to >1.6%), specifically 1% of the Werstern world (Abadie et al., 2011), and iii) gluten sensitivity, a pathology of intolerance to gluten that has been recently rediscovered (Sapone et al., 2011), which excludes CD and wheat allergy, with an estimated prevalence of 6% for the USA population. There is evidence that there are peptides that are responsible for celiac disease and gluten allergy. However, the intolerance's and non-celiac gluten sensitivity have been related to other components present in bread. Much research has been focused on determining the peptide sequence promoting bowel inflammation, although very much remains unknown. CD is an inflammatory disease of the upper small intestine caused by glutamine- and proline-rich peptides from cereal storage proteins (gluten) with a minimal length of nine amino acids, but innate immunity is also involved in the damage produced by these cereals (Bernardo et al., 2012). In people with a genetic susceptibility, such peptides are insufficiently degraded by gastrointestinal enzymes; they permeate the lymphatic tissue, are bound to celiac-specific, antigen-presenting cells, and stimulate intestinal T-cells. Till now, the only effective therapy is based on a gluten-free diet (GFD) long-life, which explains the interest in developing safe, nutritionally balanced and palatable gluten free foods. This poses challenges for the use of cereals to develop gluten free-products. Nevertheless, this challenge has been met by agronomic and food technology approaches, which will be discussed in the next sections. This review will be focused on cereal-based commodities available for developing gluten-free blends and the biochemical tools for mimicking gluten network viscoelastic properties. Special emphasis will be put on describing the available techniques for gluten detection and quantitation. 2. Gluten free cereals 2.1 Cereals reported to be gluten free Although the name wheat is always associated with gluten and with toxicity for coeliac patients, some reports have stated that some wheat cultivars are safe for this population. In fact, Spaenij-Dekking et al. (2005) investigated whether wheat varieties exist with a natural low number of T-cell-stimulatory epitopes by analysing gluten protein sequences present in public databases. In their study, wheat accessions from diploid (AA, SS/BB, and DD genomes), tetraploid (AABB), and hexaploid (AABBDD) Triticum species were also tested for the presence of T-cell-stimulatory epitopes in gliadins and glutenins by both T-cell and monoclonal antibody-based assays. The authors identified gluten proteins that lack one or more of the known T-cell-stimulatory sequences suggesting that it would be possible to select and breed wheat varieties that contain low amounts of T-cell-stimulatory sequences. One example is Triticum monococcum, the oldest and most primitive cultivated wheat. Pizzuti et al. (2006) evaluated the toxicity of this wheat by using an in vitro distal duodenum biopsies culture system, which were subjected to conventional histological examination and immunohistochemical detection. This revealed that T. monococcum gliadin shows a lack of toxicity. Cereals that are always considered gluten free are rice (Oryza sativa L.), maize (Zea mays L.) and sorghum (Sorghum bicolor (L.) Moench), which are distant relatives of wheat and are known to be safe for coeliacs. In addition, a number of species of millets, the Ethiopian cereal teff, and a range of pseudocereals are also available for providing gluten free flours, even increasing the nutritional pattern of those products in the case of pseudocereals (Alvarez- Jubete et al., 2010). Nevertheless, rice and corn are low in protein, fibre, and folate (Table 1); whereas teff, quinoa, amaranth and buckwheat show a favourable fatty acid composition and they are high in protein (Hager et al., 2012). In particular, amaranth and teff are high in calcium, magnesium and iron, although those contents are greatly dependent on the region of cultivation. Rice flour is the most suitable commodity for bakery applications due to its bland taste, white colour, digestibility and hypoallergenic properties. Other attributes such as the low content of protein and sodium and the presence of easily digested carbohydrates are additional benefits. In rice, the ratio of albumin-globulin-prolamin-glutelin is unique among the cereals, having a high concentration of glutelins and low prolamins prolamins (Hamaker, 1994). As in the other cereals, rice proteins are deficient in essential amino acid lysine, but as a consequence of the ratio of protein fractions, rice has higher content of lysine than the other cereals, which is shared by oats. It is important to note that although the amino acid pattern of the proteins could be similar in different rice varieties, significant differences have been reported in protein and starch digestibility among varieties (Acquistucci et al., 2009). Immunochemical, molecular and in vitro and in vivo studies support the conclusion that sorghum does not contain peptides that are toxic for coeliac patients. The analysis of aqueous/alcohol-soluble prolamins (kafirins) from different sorghum varieties provides molecular evidence for the absence of toxic gliadin-like peptides in sorghum (Pontieri et al., 2013). A similar study carried out with amaranth revealed that all of the amaranth samples studied showed similar binding affinities for both specific anti-gliadin antibodies and human IgAs, and the molecular characterisation of amaranth proteins suggests that it is safe for coeliacs to consume (Ballabio et al., 2011). Teff, a low risk cereal for coeliacs that is grown in Ethiopa and other countries, has an excellent amino acid composition (including all 8 essential amino acids for humans), very high fibre content and high nutrient content in general, being suitable for gluten-free applications (Gebremariam et al., 2012). 2.2 The oats controversy: selection of oat varieties with no toxicity in coeliac disease There is an on-going debate concerning the presence or absence of proteins in oats that could be toxic for coeliac patients. Traditionally, treatment with a gluten free diet GFD has excluded not only wheat, barley, and rye, but also oats. Oats, like rice, differs from other cereals in their prolamin content. The percentage of proline and glutamine (amino acids abundant in toxic regions) in avenins is lower than in prolamins from toxic cereals (Figure 2). However, there is still some debate about the safety of oats (Pulido et al., 2009). Several in vivo and in vitro studies have indicated that the majority of coeliac subjects could tolerate moderate amounts of pure oats. Some countries permit the use of oats in “gluten-free” products, e.g. Gluten Free Oats®. According to the Codex Alimentarius Commission (2008), oats can be tolerated by most, but not all, people who are gluten-intolerant. Therefore, the acceptance of oats that are not contaminated with wheat, rye, or barley in foods covered by this standard may be determined at the national level. Moreover, according to the Commission of the European Communities (2009), a major concern is the contamination of oats with wheat, rye, or barley that can occur during grain harvesting, transport, storage, and processing. Therefore, the risk of gluten contamination in products containing oats should be taken into consideration with regard to labelling of products. Some cross-reactivity with gliadin-specific antibody has been attributed to wheat contamination in oat-based food (Pulido et al., 2009). However, other authors have reported clear evidence suggesting that avenins have the ability to induce the activation of mucosal T-cells, causing gut inflammation and villous atrophy (Pulido et al., 2009). Arentz-Hansen et al. (2004) described the intestinal deterioration suffered by some CD patients following the consumption of oats while on gluten free diet. Avenins can trigger an immunological response in these patients similar to the response produced by the gluten of wheat, rye, or barley. The monitoring of 19 adult coeliac patients who consumed 50 g/day of oats over twelve weeks showed that one of the subjects was sensitive to oats. Therefore, it is critical to clarify either qualitatively or quantitatively the potential immunotoxicity of oats to coeliac patients (Arentz-Hansen et al., 2004; Pulido et al., 2009). Comino et al. (2011) showed using the anti-33-mer antibodies (G12 and A1) raised against the toxic fragment that oat immunogenicity for CD patients varies according to the cultivar. The intensity of the signal obtained with the antibody was proportional to the potential damage caused to CD patients, thereby providing a rational explanation of why some oats trigger immunological response, and a solution to avoid the presence of such varieties in gluten-free diets. 3. Transgenic approaches for developing cereals free of immunotoxic gluten The identification and/or breeding of wheat cultivars or close relatives naturally deficient in immunogenic prolamins is very appealing. However, none of the tested materials (including landraces, cultivars, and deletion lines) was completely nontoxic for CD patients (Spaenij Dekking et al., 2005; van den Broeck et al., 2009; Comino et al., 2012a). The high level of complexity of gliadin genes and the fact that they may contain multiple and different T-cell epitopes (van Herpen et al., 2006), make conventional breeding approaches to obtain wheat varieties with reduced content of immunotoxic sequences very difficult. The use of genetic engineering to down-regulate gene expression is now routinely used in many crops, including all the major cereals, and is therefore an attractive opportunity for reducing the immunotoxic components of gluten and, therefore the incidence of gluten-related allergies and intolerance in cereals. Most of the examples listed below of down-regulation of gluten proteins in wheat and other cereals are based on RNA interference (RNAi) technology (Table 1). Although not all of them were specifically aimed to develop low-gluten cereals for human allergies and intolerance, they prove that the down-regulation of the different gluten fractions related to the CD can be achieved using this technology. RNA silencing is a sequence-specific RNA degradation system that is conserved in a wide range of organisms, and termed post-transcriptional gene silencing and RNAi, in plants and animals, respectively. RNAi is a post-transcriptional process triggered by double-stranded RNA (dsRNA), which leads to gene silencing via a two-step mechanism (Watanabe, 2011). Becker et al. (2006) reported the use of hpRNA constructs to knock down the a-gliadins in cv Florida. They produced a range of transgenic plants with the -gliadins silenced. Flours from transgenic and wild type lines were analysed by RP-HPLC, showing a 63% reduction of the a-gliadins in the transgenic lines (Becker et al., 2012) (Table 1). The loss of a-gliadins was compensated by the increase of albumins/globulins (34%), -gliadins (23%), -gliadins (20%), HMW-GS (16%) and glutenin-bound -gliadins (8%). In contrast, LMW-GS were not affected. Overall, the total contents of gliadins and gluten were reduced, respectively, by 16 and 9%. Interestingly, the crude protein content was similar in transgenic lines and in the wild type. Lange et al. (2007) introduced an antisense construct against C-hordeins (the sulphur-poor storage proteins of barley which are homologous to the -gliadins of wheat) into barley cv 'Golden Promise'. The reduction of the target proteins ranged from 29 to 40% depending on the transgenic line. Other storage proteins were affected by the down-regulation of C- hordeins; the relative amount of /B-hordeins was increased in the transgenic lines while there were also minor relative increases in the amounts of D-hordeins (homologous to the HMWGS of wheat). The increase in the sulphur-rich /B-hordeins and D-hordeins was related to significant higher expression levels of the corresponding genes (Hansen et al., 2007). Interestingly, this re-distribution in the proportions of storage proteins resulted in a more balanced amino acid composition; the five antisense lines had lower amounts of proline, glutamic acid/glutamine, and phenylalanine (up to 12%, 6%, and 9% reduction), while the lysine, threonine, and methionine contents were increased by up to 16%, 13% and 11%, respectively. Gil-Humanes et al. (2008) and Piston et al. (2011) also used two hpRNA constructs to silence the -gliadins in two genotypes of the bread wheat cv ‘Bobwhite’. They reported 18 transgenic lines with reductions in the -gliadin fraction. However, the down-regulation was variable, ranging from 65 to 97% depending of the transgenic line (Piston et al., 2011). The reduction of -gliadins was also accompanied by an increase in other storage proteins, in particular, the - and -gliadins (Table 2). However, the total gliadin content did not differ significantly in the transgenic lines relative to the wild type. Likewise, the transgenic lines showed increases in the HMW-GS, LMW-GS and total glutenin contents. Overall, the gluten content was increased by an average of 50% and the total protein content was slightly higher in most of transgenic lines, but more significantly for lines of one of the genotypes. They carried out an in silico analysis of the amino acid composition of gluten proteins in transgenic lines with down-regulated -gliadins and control lines, showing that the overall proportions of amino acids were not significantly modified in the transgenic lines relative to the wild type rice loaves with very soft crumb and extended the shelf life (Gujral et al., 2003a, b). Other useful enzymes in rice bread formulation are glucose oxidase and transglutaminase (Gujral et al., 2004a, b) that catalyse the formation of inter and intramolecular crosslinks between the cereal proteins leading to a protein network (Marco and Rosell 2007, 2008). Protein crosslinking catalysed by transglutaminase was confirmed by the decrease in the amount of free amino groups and the disappearance of numerous protein bands as a consequence of the formation of large protein polymers, linked by isopeptidic and disulphide bonds, with reduced solubility (Marco et al., 2007). Numerous alternatives have been reported recently to improve nutritionally gluten free products; it is not the intention of this review to list all of them, only to point out the most significant ones. The use of whole grains mainly to increase the fibre content or the addition of bran has been proposed to improve the nutrient profile of gluten-free products (Sairam et al., 2011). Making blends of cereal and pseudocereal flours such as buckwheat flour may give to gluten-free breads with enhanced contents of total phenolic compounds, rutin and quercetin contents and antioxidant activity (Sakac et al., 2011). Pregerminated brown rice is another alternative bread ingredient because it is more nutritious after germination with increased free gamma amino butyric acid (Charoenthaikij et al., 2010; 2012). 5. Gluten analysis Gluten may trigger immune responses of different kinds in predisposed individuals. Since the only effective treatment is the avoidance of gluten containing foods, it is necessary to have reliable methods for gluten determination. Gluten proteins have unique compositions with approximately 15% proline and 35% glutamine residues (Alaedini and Green, 2005). Although most dietary proteins are digested into simple amino acids, dipeptides, and tripeptides by gastrointestinal proteases, the high content of proline residues makes the gluten peptides resistant to degradation by digestive proteases (Bethune and Khosla, 2008; Fasano, 2009). About 10% of gluten appears to be made up of potentially toxic gliadin peptides (Khosla et al., 2005), thus it is desirable to quantify the amounts of these peptides, so that the toxicity of the gluten present in foods can be established more precisely. Nevertheless, Although - and gliadin-derived peptides are immunodominant in adults, responses to the LMW glutenins and -gliadins have been frequently observed in children and occasionally in adults (van de Wal et al., 1999; Vader et al, 2002). The presence of the numerous gluten components, the variation in the extraction efficiency, and the lack of reference materials are some issues that hinder the implementation of equivalent laws at national level and the comparison of data across the different methods (Diaz-Amigo et al., 2012). 5.1. Factors affecting gluten analysis Proteins are modified to improve their functionally and increase their usage in different applications to form and stabilise structures. Proteins can be modified in various ways as deamidation, transamidation and degradation. All of these modifications can also happen naturally due to enzymes in cereal seeds. Hydrolysis is widely used in the preparation of wheat products in the food industry. Hydrolysis may decrease the toxicity of gluten (Greco et al., 2011) but also the fragmentation of peptides can make it more difficult to analyse gluten in these foods (Mena et al., 2012). Deamidation of gluten proteins is a widely used process in the food industry that leads to increased charge density on the protein, causing changes in protein conformation due to electrostatic repulsion. The analysis of deamidated gluten is more difficult as there is a decrease in the affinity and recognition of antibodies to gluten proteins and peptides, which may lead to underestimation when immunoassays are used to the quantify gluten content of foods (Kanerva et al., 2011). An important factor in prolamin and glutelin analysis is complete extraction from the samples. The extraction of gluten from wheat, barley, rye and oat standards is easier than from processed foods. During the processing of some foods, proteins are treated at high temperatures in a dry state at a neutral pH, forming isopeptide bonds between the -amino groups of lysine and the - or -carboxamide groups of asparagine and glutamine residues. Furthermore, the heat-treatment of cooked and baked products leads to the formation of protein aggregates in an insoluble matrix that makes analyses even more difficult. Therefore it is necessary to use an extraction system giving complete recovery of both prolamins and glutelins, to guarantee that products marketed as suitable for coeliacs are really gluten-free. The so-called cocktail solution combines reducing and disaggregating agents to extract gluten proteins completely as this ensures that protein aggregates are disrupted. (Garcia et al., 2005). However, it is not compatible with all the techniques used for gluten analysis because - mercaptoethanol interferes with the specific binding of the antibodies used in some types of immunoassays, giving false results. More recently, another combination of reagents called UPEX (universal prolamin and glutelin extractant solution) leads to a complete extraction and it is compatible with all gluten analysis procedures (Mena et al., 2012). This solution includes the odourless reducing agent, Tris (2-carboxyethyl)-phosphine (TCEP) that is more specific for breaking disulphide bridges and less toxic than the other reducing agents commonly used (Han, 1999) and the disaggregating agent N-Lauroylsarcosine, widely used in plant cell lysis, which contributes to opening polypeptide chains and is even more efficient than guanidine hydrochloride (Schlink and Reski, 2002). In addition, a novel universal gluten extraction solution (UGES) has recently been described (Biomedal Diagnostics, Seville, Spain). The components of this gluten extraction solution are a reducing agent, a solubilising agent (arginine) and an antiseptic agent in ethanolic solution (patent WO 201231612; Muñoz-Ruano et al., manuscript in preparation). The UGES procedure gave high extraction efficiency from both simple and complex matrices even if they had heat-processed. Another of the critical points in gluten analysis is the use of a correct standard. This standard should be as representative as possible of the gluten proteins to be analysed. The Working Group on Prolamin Analysis and Toxicity (PWG) gliadin standard is the standard most used internationally for gluten analysis. This standard is obtained from a mixture of 28 wheat cultivars representative of the European wheat-producing countries (van Eckert et al., 2006). Nevertheless, cereals contain a greater number of proteins than those present in the PWG standard. Some authors have suggested that it would be more correct to use a hydrolysed standard combined with a competitive assay to quantify peptides of partially hydrolysed gluten in fermented wheat, rye, and barley products (Gessendorfer et al., 2009). Commercial foods usually have only partial hydrolysis and, when the proteins are exhaustively hydrolysed, the toxicity for coeliac patients of the peptides generated usually disappears (Stepniak et al., 2006). Comparing the intact PWG gliadin standard with a partially enzimatically digested gliadin standard it has been demonstrated that the resulting curves are similar in each case when applying a competitive immunoassay and therefore the intact PWG gliadin standard can be used as a more accessible gold standard as it is more difficult to perform a reproducible hydrolysed standard (Mena et al., 2012). However, other strategies based on the use of immunotoxic peptides of gluten as standard are being developed for the analysis of samples with gluten hydrolysate This standard presented a high degree of repeatability, reproducibility and stability and the results obtained were correlated with the potential relative immunotoxicity of gluten (Comino et al., 2012b; 2013)”. 5.2. Immunological techniques Many methods for detecting prolamin proteins have been developed over the years. The complexity of prolamin proteins, however, creates an enormous challenge in accurately quantifying the total prolamin content. Some methods including the polymerase chain reaction (PCR), HPLC and MS, may be used simultaneously with immunological methods. One of the most recent methods developed for gluten analysis is a potentiometric electronic tongue, which was able to detect 1-2 mg/Kg of gliadin from different matrices (Peres et al., 2011). The most practical methods for food analysis is based on immunological analysis. An ideal antibody for gluten analysis in foods should be not only a reliable indicator of the presence of prolamins from cereal species known to be toxic to CD patients but also should recognise the specific intramolecular regions responsible for such toxicity. Nevertheless, there are many such regions and even today not all have been identified. 5.2.1. Antibodies testing for gluten-free foods Several antibodies (Ab) have been raised against different prolamin epitopes. The anti - gliadin Ab is used in a sandwich format that was approved as an official method by AOAC (Association of Official Agricultural Chemist, Skerrit and Hill, 1990, 1991). Other antibodies were raised against different epitopes of -gliadin, such as PN3 (residues 31-49) for the toxic 19-mer peptides (Sturgess et al., 1994; Ellis et al., 1998), CD5 (residues 51-75), Abs against T-cell stimulatory peptides present in gluten (Mitea et al., 2008), and R5 which recognises highly repeated peptide sequences present in wheat, barley and rye grains. This antibody is a monoclonal antibody (Valdés et al., 2003; Mendez et al., 2005) that reacts with the epitope QQPFP and other motifs such as QLPFP, LQPFP, and QQQFP present in coeliac-toxic sequences that occur repeatedly in gliadins, hordeins, and secalins being highly sensitive towards these prolamins (Valdes et al., 2003). Recent reports recommend updating the concept of “gluten detection” to “potential relative immunotoxicity of gluten” for the safety of coeliac consumers. Two monoclonal antibodies, G12 and A1 (anti-3-mer antibodies), were raised against the main immunogenic epitope of the -gliadin 33-mer. These antibodies were able to detect the presence of gliadin 33-mer-related epitopes in prolamins from wheat, barley, rye and various oats varieties (Morón et al., 2008a, b; Comino et al., 2011), and G12is a reliable tool for detecting oat varieties potentially safe for patients with celiac disease. These antibodies have proved to be very useful for the detection of toxic peptides in food samples and for the enzymatic detoxification of gluten in clinical research (Shan et al., 2002; Morón et al., 2008a; 2008b; Ehren et al., 2009). 5.2.2. Techniques A standardised method of analysis is required to quantitatively determine the gluten content of food and provide the basis for enforcing regulations controlling the use of the term “gluten free” in food labelling. People with CD should feel confident that foods labelled “gluten-free” have been analysed for gluten using the “the best available” methodology. In January 2009, the European Commission published a new European Regulation concerning the composition and labelling of foodstuffs suitable for people intolerant to gluten; this regulation states that foods may display the term “gluten-free” if the gluten content does not exceed 20 mg/kg as sold to the final consumer (Commission of the European Communities, 2009). Food containing between 20 ppm and 100 ppm of gluten is considered to be very low in gluten content according to the EU laws, but there is no full agreement among countries about the term “gluten-free food”. Consequently, methods for gluten analysis must be sensitive enough to quantify these levels of gluten in foods. 5.2.2.1. Enzyme-linked immunosorbent assays (ELISAs) ELISAs have become the recommended methods in gluten analysis. Immunological methods are based on the antibodies raise against the different prolamin fractions or specific sequences found in prolamins. The requirement for the assays is that they should measure the harmful proteins and peptides, regardless of the type of food or manufacturing process (Denery-Papini et al., 1999). The sandwich R5 ELISA is the most common enzyme immunoassay formats used in detection of gluten proteins. It is highly sensitive and is especially useful for the quantification of antigens when their concentration is low, when they are contained in samples with a large amount of contaminating proteins, or both. This assay uses two antibodies that bind to different sites on the antigen. Nowadays, it is the usual method for determining gluten content in gluten-free foods internationally accepted by the Codex Alimentarius Commission (Codex Alimentarius Commission, 2006) combined with what is known as the cocktail extraction solution. This ELISA is based on the R5 antibody and has a limit of quantification of 1.56 ppm of gliadins (Valdes et al., 2003). In hydrolysed foods the quantification of gluten by the sandwich R5 ELISA would yield lower values than the true gluten content. A sandwich ELISA using the monoclonal G12 and A1 antibodies gave very promising results for gluten analysis across a range of samples (Morón et al., 2008a; Halbmayr-Jech et al., 2012). This method had a detection limit for wheat, barley, and rye prolamins of <1ppm. Furthermore, the reactivities of these antibodies were correlated with the potential immunotoxicity of those dietary grains from which the proteins were extracted, thereby providing a rational explanation for why some cereal varieties trigger immunological response, and enabling the presence of such varieties to be avoided in gluten-free diet (Comino et al., 2012 a). The competitive R5 ELISA, based on the R5 monoclonal antibody, leads to an accurate quantification of both intact and fragmented gluten because it uses only one antibody and requires only one epitope. In addition, the competitive system is cheaper and faster than the sandwich system ELISA (Mena et al., 2012). The Codex Alimentarius Commission states that a modification of the R5 assay (competitive ELISA) has to be applied for the detection of hydrolysed gluten (Codex Alimentarius Commission, 2006). However, the Food and Drug Administration (FDA) comments that analysts should be free to use any method that provides comparable results and that other methods may be equivalent. Another comment urged FDA to remain flexible as to the method of test validation and added that not specifying analytical methods would permit a more rapid development of dependable and affordable technologies for testing gluten (Federal Register / Vol. 78, No. 150 / Monday, August 5, 2013 / Rules and Regulations). The competitive technique is not compatible with the cocktail extraction solution, but the combination of this technique with the UPEX solution leads to complete gluten analysis. The limits of detection and quantification of the competitive R5 ELISA are 0.36 and 1.22 ng/ml of gliadins, respectively, being lower in liquid samples. Recently, a collaborative study has confirmed that the two R5 antibody-based ELISA test kits are able to detect gliadin at the lower level of the limit of detection with good reproducibility and repeatability (Immer and Haas-Lauterbach, 2012). A competitive ELISA method was also developed for the detection of toxic gluten peptides in hydrolysed foods based on G12 antibody. This assay is highly sensitive and reproducible with a detection limit of 0.44 ppm gliadin. This ELISA system showed high reproducibility and repeatability (Morón et al., 2008b; Ehren et al., 2009). 5.2.2.2. Western blot Single and two-dimensional gel electrophoresis (both SDS-PAGE and A-PAGE) have been employed by different authors in order to characterise wheat, barley and rye proteins (Shuaib et al., 2007; Salmanowicz and Nowak, 2009). Nevertheless, these techniques have not enough sensibility for quantifying gluten in gluten free foods. In order to solve this problem, the separated proteins after one-dimensional SDS-PAGE are electrotransferred onto a polyvinylidene difluoride membrane where the proteins are adsorbed. A specific antibody is the added, such as the R5 antibody (Garcia et al., 2005), G12 antibody (Morón et al., 2008a, b) or anti cells T gliadin alpha-20 antibody (van den Broeck et al., 2009). These immunological western blot techniques can be used for the confirmation of gluten content in foods. 5.3. Non-immunological techniques The quantitative analysis of prolamins is mainly based on immunological methods, but mass spectrometric and chromatographic techniques have also been used (Wieser et al., 1998; Sealy-Voyksner et al., 2010). The use of complementary and alternative non-immunological systems to confirm the results of the immunological methods is very important especially in certain complex foods. 5.3.1. Proteomic techniques Applying proteomics to this field is of great interest to complement other techniques but the analysis of gluten-free foods, where gluten proteins are in low amount comparing with the other major proteins presents more difficulties (Mamone et al., 2011). Mass spectrometry (MS) is one of the more important physical methods used nowadays due to its high sensibility, for identification, characterisation and quantification of proteins and peptides. The bases of these methods are an ionisation of molecules to study, separation of ions in basis to their relation mass/charge and detection of the separated ions. Depending on the different method of ionisation, separation and detection, there are several MS techniques, used in different applications. MALDI-TOF MS was the first technique used to identify toxic prolamins involved in coeliac disease. Even though MALDI-TOF gluten analysis is very useful (Hernando et al., 2003), identifying gluten based on the analysis of intact proteins is not sufficient, due to extensive sequence similarities among gluten proteins; the results for hydrolysed gluten are also insufficiently accurate. For unambiguous gluten protein identification, a proteomic approach involving tandem mass spectrometry (MS/MS) or multistage MS experiments would be very promising. 5.3.2. Quantitative real-time PCR (Q-PCR) Several polymerase chain reaction (PCR) based methods for the detection and quantification of DNA of gluten-containing cereals have been described, but there are very few studies of their application to the analysis of gluten in foods. 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