Abstract
Food-induced allergies are considered an important problem of public health with special impact in the quality of life of the sensitised/allergic individuals. As highly consumed foods, fish and shellfish represent a valuable source of proteins for the general population. In spite of their economical and nutritional importance, these foods are known to induce hypersensitivity reactions in sensitised/allergic individuals. So far, parvalbumins (fish) and tropomyosins (crustaceans and molluscs) have been considered major allergens in seafood allergy, being responsible for most of the reported cases of adverse immunological responses. More recently, other proteins such as arginine kinases, myosin light chains, troponins and sarcoplasmic calcium-binding proteins have been regarded as relevant allergens in fish, crustaceans and molluscs. This review focuses on seafood allergens, reporting an updated and compiled list of allergens from fish, crustaceans and mollusc species, with an overview on the most representative analytical methods for their detection.
Introduction
Seafood plays an important role in human nutrition and health, which is considered an excellent source of highly assimilated proteins, vitamins and polyunsaturated fatty acids, such as docosahexaenoic acid (22:6) and eicosapentaenoic acid (20:5) from the n-3 series (omega-3) (Perez-Gordo et al., Citation2011; Sharp & Lopata, Citation2014). There is a solid scientific background reporting the wide range of health benefits associated with the ingestion of omega-3 fatty acids, such as the prevention of cardiovascular diseases and cancer or the improvement of glycaemic control (Larsen, Eilertsen, & Elvevoll, Citation2011; Mozaffarian, Bryson, Lemaitre, Siscovick, & Burke, Citation2005; Sirot, Oseredczuk, Bemrah-Aouachria, Volatier, & Leblanc, Citation2008; Van Do, Elsayed, Florvaag, Hordvik, & Endresen, Citation2005).
Due to the referred well-established health benefits, the consumption of fish and shellfish has been continuously increasing worldwide. For the majority of the world’s population, the growing interest in seafood intake can be considered a nutritional advantage. However, for a small but rather significant part of food-allergic individuals, the consumption of products containing undeclared seafood can pose severe health problems (e.g. systemic immunological reactions, anaphylaxis) as result of accidental exposure to the offending food (Madsen et al., Citation2012). In the recent years, more cases of fish and shellfish allergies have been frequently reported, being currently viewed as an emergent issue of public health. The clinical diagnosis of specific food allergies, such as seafood allergy, is based on self-reported symptoms (clinical history), specific immunoglobulin E (sIgE) blood tests or skin prick test (SPT) sensitisation, rather than open food challenges (OFC) or double-blind placebo-controlled food challenges (Burks et al., Citation2012), making the true prevalence of seafood allergy difficult to establish. In spite of this, recent data seem to suggest that 0.1–0.4% of general population is affected by fish allergy, while over 2% suffers from shellfish allergy. Nevertheless, the referred prevalence can vary with specific geographical and cultural eating habits and/or with the type of food processing (Chen, Cao, et al., Citation2013; Kamath et al., Citation2014; Kuehn et al., Citation2013). In the USA, the available reports suggest that 0.4% of the population suffers from fish allergy and 0.2% is affected by both fish and shellfish allergies. In Europe, fish allergy was estimated to have an overall incidence of 0.2%, while the allergy to shrimp, a major contributor in shellfish allergy, presented a prevalence of 5.4% (Burney et al., Citation2010; Sicherer, Muñoz-Furlong, & Sampson, Citation2004). Coastal countries like Portugal and Finland, where the consumption of seafood is very high, were not included in the referred European prevalence study. Therefore, the prevalence of seafood allergy in Europe is probably underestimated (Lopata, O’Hehir, & Lehrer, Citation2010; Perez-Gordo et al., Citation2011; Sharp & Lopata, Citation2014; Tsabouri et al., Citation2012).
Fish and crustaceans are known to induce hypersensitivity reactions mediated by the IgE in sensitised/allergic individuals (Kuehn, Scheuermann, Hilger, & Hentges, Citation2010; Lee & Taylor, Citation2011), being two of the eight groups responsible for almost 90% of worldwide reported food allergies (CODEX STAN 1-Citation1985). The routes of exposure for seafood allergy are ingestion, direct contact (skin) or inhalation of their odours or fumes created during preparation/cooking of derived foods (Kuehn et al., Citation2010; Lee & Taylor, Citation2011). For allergic individuals, the only effective means of preventing an adverse reaction is the total avoidance of seafood or the use of a therapeutic treatment (e.g. antihistaminic, corticosteroids, epinephrine) in the case of an accidental exposure to the allergenic food (van Hengel, Citation2011). Consequently, it became imperative to improve consumer’s protection through an accurate food labelling system, in order to prevent potential life-threatening risks for sensitised/allergic individuals (Costa, Carrapatoso, Oliveira, & Mafra, Citation2014; Rencova, Kostelnikova, & Tremlova, Citation2013). According to the recent European Union (EU) regulations, food producers are obligated to declare the presence of 14 groups of foods that are recognised as potentially allergenic, namely fish, crustaceans, molluscs, celery, mustard, sesame seed, gluten, tree nuts, peanuts, milk, eggs, soybeans, lupine and sulphites, as well as highlighting them from the rest of the list of ingredients (Directive 2000/Citation13/EC; Directive Citation2007/68/EC; Regulation (EU) No 1169/2011).
This report intends to provide a general and updated overview on seafood allergens, focusing on fish and shellfish (crustaceans and molluscs), the main consumed seafood groups, including a brief description of the most representative analytical methods for their detection.
Fish allergens
So far, some families of proteins such as enolases, aldolases and parvalbumins have been classified as allergens in fish, although the most representative one corresponds to parvalbumins. Included in the calcium-binding proteins, which comprise the second most important family of animal food allergens, parvalbumins are currently reported as responsible for more than 95% of food allergies induced by fish. Generally, the symptoms occur within 30 min after the contact with the offending food and can result in skin, respiratory and gastrointestinal symptoms, including less frequent fatal systemic responses such as anaphylaxis (Kuehn et al., Citation2010; Lee & Taylor, Citation2011; Weber & Paschke, Citation2010).
Parvalbumins
Parvalbumins are small (10–13 kDa), acidic and water-soluble proteins, presenting remarkable resistance to high temperatures, denaturing agents and proteolytic activity (Griesmeier et al., Citation2010; Lee & Taylor, Citation2011; Weber & Paschke, Citation2010). They are usually divided into two evolutionary lineages of isoforms: the α-parvalbumins, which are generally classified as non-allergenic, and the β-parvalbumins, wherein the majority of IgE-reactive parvalbumins are included (Jenkins, Breiteneder, & Mills, Citation2007; Wang et al., Citation2014; Weber & Paschke, Citation2010). Parvalbumins are abundant proteins in the white muscle of many fish species, performing an important role in the relaxation of muscle fibres through the binding of free intracellular calcium. They are composed by two functional domains, each binding a calcium ion, and a third silent domain protecting the hydrophobic core of the protein. In these proteins, the binding of calcium is thought to be of critical relevance to the integrity of the conformation of the IgE epitopes. Calcium depletion is known to induce structural alterations in these proteins, decreasing the allergenic capacity of parvalbumins (Arif, Jabeen, & Hasnain, Citation2007; Bugajska-Schretter et al., Citation1998; Bugajska-Schretter et al., Citation2000; Capony & Pechère, Citation1973). These sarcoplasmatic proteins are present at high proportion in the bottom dwelling fish species, such as cod, whiff or flounder. Therefore, these species are expected to have higher allergenicity than active fishes (rich in dark muscle) such as tuna, mackerel, swordfish and skipjack (Jenkins et al., Citation2007; Rencova et al., Citation2013; Tsabouri et al., Citation2012).
Tolerance to a certain fish species can vary greatly among allergic individuals, so there is about 50% of chance for a sensitised patient to be cross-reactive to more than one fish species. This happens because the secondary and tertiary structures of parvalbumins are highly conserved, though their amino acid sequences (primary structures) can differ substantially among fish species (Sharp & Lopata, Citation2014). In spite of evidencing very distinct IgE-binding epitopes, limited data on epitope alignment of four parvalbumins from different fish species, namely salmon, cod, mackerel and carp, seem to indicate the presence of a highly antigenic region (region IV; Bugajska-Schretter et al., Citation1998; Perez-Gordo et al., Citation2012; Sharp & Lopata, Citation2014), which might be responsible for the polysensitisation to multiple fish species in allergic individuals (Griesmeier et al., Citation2010). Current studies on cross-reactivity phenomena highlight the need of sensitised/allergic individuals to eliminate from diet any kind of fish, even before the performance of allergy diagnosis by SPT, serum-specific IgE blood tests or OFC (Carrapatoso, Citation2004; Lieberman & Sicherer, Citation2011; Muraro et al., Citation2014; Perez-Gordo et al., Citation2011; Sharp & Lopata, Citation2014; Tsabouri et al., Citation2012).
Another major aspect of fish allergy concerns the fate of allergenic proteins during food processing since parvalbumins are considered highly stable proteins, being most frequently resistant to common physical and chemical processes. So far, it is still uncertain how food processing may affect parvalbumins from distinct fish species. According to literature, heat treatments do not affect IgE-binding capacity since these proteins are able to return to their original conformation after cooling (Mills, Sancho, Rigby, Jenkins, & Mackie, Citation2009). IgE-binding capacity of parvalbumins can be reduced by chemical processes. Proteolysis, often combined with pH alterations, is another efficient way of decreasing allergenicity; however, it may also contribute to expose pre-existing epitopes or create new epitopes by aggregation (Sletten, Van Do, Lindvik, Egaas, & Florvaag, Citation2010; Thomas et al., Citation2007).
Codfish allergy is presently the best well studied since most fish-allergic patients do not tolerate cod. The major allergen designated as Gad c 1, which is isolated from Baltic cod (Gadus callarias), is often used as a reference molecule for the study of parvalbumins. Other homologous allergens have been isolated from worldwide highly appreciated commercial fishes, namely other cod species (Gadus morhua), common carp (Cyprinus carpio), Atlantic salmon (Salmo salar), Japanese jack mackerel (Trachurus japonicas), bigeye tuna (Thunnus obesus) and European hake (Merluccius merluccius; Kuehn et al., Citation2010; Perez-Gordo et al., Citation2011; Tsabouri et al., Citation2012).
In recent years, the number of identified allergenic proteins available at databases has increased, improving the establishment of evolutionary and structural relationships among allergens from distinct origins (Radauer, Bublin, Wagner, Mari, & Breiteneder, Citation2008). Allergen platforms such as the Official List of Allergens issued by the International Union of Immunological Societies (IUIS) Allergen Nomenclature Sub-committee and the ALLERGOME database have become excellent tools for allergen classification since they report molecular, biochemical and clinical data about allergenic proteins. and summarise all fish allergens that were already characterised, being currently available at databases (ALLERGEN, Citation2014; ALLERGOME, Citation2014). Most of the identified fish allergens correspond to parvalbumins, which account for more than 200 entries (), although different proteins, namely enolases and aldolases (), are also defined as IgE-reactive in fish species.
Table 1. List of fish allergens (parvalbumins) and corresponding fish species.
Table 2. List of fish allergens (enolases and aldolases) and corresponding fish species.
Enolases and aldolases
With respect to other families of allergenic proteins in fish (), 50-kDa enolases and 40-kDa aldolases have been recently described as important allergens in highly consumed fishes such as cod, salmon and tuna (Kuehn, Swoboda, Arumugam, Hilger, & Hentges, Citation2014; Kuehn et al., Citation2013). Both enzymes have biological functions in metabolic glycolysis, being involved in the sugar degradation for the production of energy (Kuehn et al., Citation2014). The biochemical characterisation of enolases seems to indicate that they are dimeric proteins, while aldolases present oligomeric profiles. Additionally, no post-translational modifications such as glycosylation and/or phosphorylation have been found in fish enolases and aldolases (Kuehn et al., Citation2013). Preliminary results revealed limited inter-species cross-reactivity, with fish enolases being more cross-reactive than aldolases. Both enzymes have been described as heat-labile since thermal treatments above 90°C for 1–5 minutes seem to destroy their tri-dimensional structures. Despite eliminating some conformational allergenic epitopes, new linear epitopic regions can be created during food processing, which might contribute to increase their potential allergenicity (Kuehn et al., Citation2013).
In addition to the parvalbumins, enolases and aldolases, collagen has also been pointed out as an allergen in fish. However, despite the existence of some positive in vitro allergy tests, the potential risk of fish gelatine (collagen) for triggering an adverse immunological reaction among fish-allergic individuals remains unclear (Lee & Taylor, Citation2011; Weber & Paschke, Citation2010).
Shellfish allergens
The term shellfish is a non-taxonomic designation usually used in the context of seafood consumption. This group comprises crustaceans and molluscs, representing a significant market niche of marine species with commercial interest. Crustaceans are classified as arthropods and include over 50,000 living species (shrimp, prawns, lobster, crayfish and barnacles). A large number of crustacean species are consumed either raw or cooked/processed. Molluscs are subdivided into classes of bivalves, gastropods and cephalopods, comprising almost 100,000 different species (mussels, oysters, abalone and squids). The nutritional value and intrinsic organoleptic characteristics of molluscs make them highly appreciated and consumed foods all over the world, especially in coastal regions (Kamath et al., Citation2014; Lopata et al., Citation2010; Mao et al., Citation2013).
Hypersensitivity reactions to seafood are normally immediate (approximately 30 minutes) up to 2 hours after its ingestion. Late-phase immunological responses are also possible to occur, when symptoms are developed up to 8 hours (Wang & Sampson, Citation2007). Clinical manifestations of shellfish allergy are very similar to fish allergy, resulting, not only from the ingestion of the offending food, but also from manipulating or inhaling the cooking vapours during food processing. Commonly, symptoms begin within minutes and may include oral allergy syndrome and cutaneous (urticaria, angioedema), gastrointestinal (vomiting, abdominal pains) and/or respiratory symptoms. Although less frequent, severe and systemic responses such as anaphylactic shocks may also occur upon shellfish consumption (Carrapatoso, Citation2004; Lopata et al., Citation2010; Yu et al., Citation2011).
Tropomyosins
In crustaceans and molluscs (–), different proteins are known to trigger observable clinical symptoms, although the majority of them is attributed to a family of proteins designated as tropomyosins, which are closely related alpha helical coiled-coil secondary structure proteins. Tropomyosins are present in muscle and non-muscle cells, and together with actin and myosin, they intervene in the regulatory process of muscle contraction (Breiteneder & Mills, Citation2005; Leung et al., Citation2014). So far, a great number of allergenic tropomyosins have already been described among crustaceans (), namely in shrimp, crab, lobster and prawn, whose cross-reactivity is related to the high similarity in amino acid sequences among distinct species (Leung et al., Citation2014). The homology among tropomyosins is so high that most shellfish-allergic individuals cross-react upon the ingestion of other crustacean or mollusc species. In fact, it is estimated that 75% of the individuals that present allergy to some type of shellfish is at risk of cross-reacting to a second species due to the high structural similarity among tropomyosins (Emoto, Ishizaki, & Shiomi, Citation2009; Lee & Taylor, Citation2011; Tsabouri et al., Citation2012; Weber & Paschke, Citation2010).With molecular weights ranging from 34 to 38 kDa, tropomyosins are considered heat-stable proteins. Upon food processing, these allergens can unfold at a limited extent during heating process, being able to return to their conformational structure after cooling. Chemical processes such as Maillard modification may potentiate tropomyosin allergenicity (Mills et al., Citation2009).
Table 3. List of crustacean allergens (tropomyosin) and corresponding species.
Table 4. List of crustacean allergens (other non-tropomyosin) and corresponding species.
Table 5. List of mollusc allergens and corresponding species.
Arginine kinase
In recent years, other proteins such as arginine kinases have been reported as new allergens in crustaceans and molluscs ( and ). This enzyme is a 40-kDa water-soluble protein present in myosinogen, which is involved in cell metabolism of invertebrates (Chen, Mao, et al., Citation2013). Preliminary reports on identified allergenic arginine kinases from crustaceans seem to indicate that these enzymes are unstable at temperatures between 40 and 80°C, being partially unfolded and revealing novel hidden epitopes that may be responsible for increasing IgE reactivity. Above 80°C, arginine kinase is thought to fully unfold and subsequently decrease its immunogenicity (Chen, Mao, et al., Citation2013; Giuffrida, Villalta, Mistrello, Amato, & Asero, Citation2014). Similarly to arginine kinase from crustaceans, the allergenic arginine kinase from molluscs (e.g. Oct f 2) is also unstable above 40°C, though literature suggests that its allergenic properties are reduced at lower temperatures (>48°C; Shen et al., Citation2012).
Sarcoplasmic calcium-binding and troponin C proteins
The sarcoplasmic calcium-binding and the troponin C proteins belong to the superfamily of EF-hand proteins. The sarcoplasmic calcium-binding proteins are present in the invertebrates, being considered the equivalent of the vertebrate parvalbumins that contribute to maintain the intracellular calcium (Gao, Gillen, & Wheatly, Citation2006). Due to this function, they are designated as calcium buffers, being acidic cytosolic proteins (20–22 kDa) with four potential EF-hand calcium-binding sites, from which two or three are functional (Hermann & Cox, Citation1995). Troponin C is a calcium sensor/modulator protein that regulates downstream target proteins in a calcium-dependent manner. So far, 5 troponin C and 26 sarcoplasmic calcium-binding proteins have been identified as allergens in crustaceans (), although their study is still at a very preliminary stage.
Myosin light chain proteins
Myosins are part of a complex that involves other proteins such as actin, tropomyosin and troponin, being all fundamental to muscle contraction. Myosin is composed of two heavy chains and four light chains. Each myosin heavy chain is surrounded by two light chains with 20 kDa each (Ayuso et al., Citation2008). Until now, six myosin light chains have been classified as allergens among crustaceans (). Myosin light chain allergens seem to present high IgE reactivity with sera from shellfish-allergic patients, being potentially considered as major allergens similarly to tropomyosins. They are also resistant to heat since they tend to maintain IgE-binding capacity upon processing (100°C for 5 minutes; Ayuso et al., Citation2008).
Detection of seafood allergens
The presence of trace amounts of undeclared allergenic ingredients in seafood products poses a significant health risk to sensitised/allergic individuals of suffering abnormal immune episodes as consequence of accidental exposure (Lee & Taylor, Citation2011; van Hengel, Citation2011). Considering the severity and the frequency of cases involving seafood allergy, in addition with the low levels (3–32 mg of allergenic proteins from fish or shellfish, respectively) responsible for eliciting observable symptoms upon ingestion, the sensitised/allergic individuals are obliged to completely avoid products susceptible of containing seafood as ingredient (Reese et al., Citation2005; Untersmayr et al., Citation2007). Therefore, in order to protect these patients against the presence of hidden allergens as a result of cross-contamination or mislabelling, reliable and highly sensitive analytical methods are vital to verify labelling compliance and to help the industrial management of fish and shellfish allergens (Costa, Mafra, Carrapatoso, & Oliveira, Citation2012; Herrero, Vieites, & Espiñeira, Citation2014; Lee & Taylor, Citation2011). It is generally accepted that the ideal limit of detection (LOD) for allergens in food products should range between 1 and 100 mg/kg (Poms, Klein, & Anklam, Citation2004).
So far, several molecular tools targeting either proteins or DNA have been published for food allergen analysis, namely enzyme-linked immunosorbent assays (ELISA), lateral flow devices (LFD), liquid chromatography (LC)-coupled with mass spectrometry (MS), polymerase chain reaction (PCR), real-time PCR, microarrays or biosensors. However, the potential number of available methods within the sphere of seafood allergen detection is still limited (Pascoal et al., Citation2011; Rencova et al., Citation2013).
Protein-based methods
Protein-based methods, such as ELISA or LFD, test for the presence of allergens or specific food marker proteins by using specific mono- or polyclonal antibodies that are usually raised in animals (Schubert-Ullrich et al., Citation2009). These immunochemical assays are particularly useful in food industry since they offer high specificity and sensitivity (antigen/antibody interaction) for fast allergen detection, without requiring extensive sample preparation, expensive equipment or experienced personnel. In the specific case of fish allergens, the immunochemical assays targeting parvalbumins are among the most widely used methods for their detection in foods. As previously mentioned, important variations in parvalbumin levels can be found in some of the most consumed fish species (carp, cod, hake, salmon and tuna) as highlighted in several reports (ALLERGEN, Citation2014; ALLERGOME, Citation2014; Griesmeier et al., Citation2010; Perez-Gordo et al., Citation2011; Tsabouri et al., Citation2012; Van Do et al., Citation2005). Due to the high prevalence of shellfish allergy, namely to crustacean species, the development of rapid methods for the detection of trace amounts of shellfish has become imperative. Despite some lack of available techniques to detect mollusc allergens, immunochemical methods have been the first choice for qualitative and quantitative analysis of shellfish allergens, such as the tropomyosins Cra c 1, Hom a 1, Pen m 1, Tod p 1 and Scy pa 1 (ALLERGEN, Citation2014; ALLERGOME, Citation2014; Emoto et al., Citation2009; Kamath et al., Citation2014; Yu et al., Citation2011). In spite of the simplicity and utility of the immunoassays, they also present some major drawbacks and, consequently, the results from their application should be carefully analysed. Both ELISA and LFD are prone to cross-reactivity phenomena and they are also highly affected by conformational changes in proteins upon food processing, which can lead to the possibility of false negative or positive results (Costa, Ansari, Mafra, Oliveira, & Baumgartner, Citation2014; Lee & Taylor, Citation2011).
MS methodologies have also found application in allergenomics, allowing protein identification, characterisation and quantification. The main advantages of these methods rely on their high accuracy, sensitivity, specificity and reproducibility, though the high cost of analysis represents an important drawback (Picariello, Mamone, Addeo, & Ferranti, Citation2011). Additionally, MS methods can overcome the problems of cross-reactivity phenomena often linked to immunoassays, allowing the unequivocal identification of the tested allergens/peptides. The analysis of allergens by MS methodology can be performed by one of two approaches, either targeting intact proteins (analyte and reference standards) or peptides obtained from protein digestion using proteolytic enzymes (Picariello et al., Citation2011). This methodology was already applied for the direct detection of parvalbumin peptide biomarkers using a set of 16 species of fish that were analysed by LC-MS/MS approach (Carrera, Cañas, & Gallardo, Citation2012).
Currently, there are several ELISA kits commercially available that enable the detection of fish and shellfish in foods. For example, the “Fish Protein ELISA kit” (Elution Technologies, Vermont, USA) described as capable of directly detecting parvalbumin in different food matrices has a limit of quantification (LOQ) of 1 mg/kg. The “AgraQuant® Fish” (Romer Labs Division Holding GmbH, Austria) refers a LOQ of 4–100 mg/kg, though without clear information regarding the target class of fish proteins. The “Crustacean Protein ELISA kit” (Elution Technologies, Vermont, USA) and “AgraQuant® ELISA Crustacea” (Romer Labs Division Holding GmbH, Austria) claim LOQ of 2 mg/kg and 20–400 mg/kg, respectively. “Mollusk ELISA kit” (Elution Technologies, Vermont, USA) allows the detection of mollusk proteins from 1 mg/kg.
The limits of detection (LOD) of the most relevant reports from literature on protein-based techniques applied to seafood detection range from 0.046 to 18.7 mg/kg (Carrera, Cañas, & Gallardo, Citation2012; Faeste & Plassen, Citation2008; Kuehn et al., Citation2010; Weber, Steinhart, & Paschke, Citation2009).
DNA-based methods
Technologies based on DNA analysis, namely PCR, present some advantages over the methodologies targeting proteins. DNA molecules are more stable and resistant to thermal treatments, pH alterations and partial hydrolysis than proteins, being less affected by processes that normally alter the integrity of proteins. In fact, DNA-based methods are notably helpful on the analysis of highly processed foodstuffs (Eischeid, Kim, & Kasko, Citation2013; Herrero et al., Citation2014; Hildebrandt & Garber, Citation2010; Lee, Nordlee, Koppelman, Baumert, & Taylor, Citation2012). PCR, preceded by DNA extraction, provides a sensitive tool for the specific detection of genomic sequences encoding allergenic proteins or species-specific markers. However, the amplification of a DNA sequence by PCR does not necessarily indicate the presence of an allergenic protein in the food matrix, being therefore considered an indirect method of allergen detection (Costa et al., Citation2014; Mafra, Ferreira, & Oliveira, Citation2008). There are already two commercial kits available for fish and mollusc DNA detection: “SureFood® ALLERGEN ID Fish” and “SureFood® ALLERGEN ID Molluscs” (R-Biopharm AG Darmstadt, Germany; LOD ≤ 0.4 mg/kg).To our knowledge, the majority of studies in the literature reporting the application of PCR-based methodologies for fish analysis have mainly been focused on species authentication purposes. However, there are some studies regarding the use of PCR and real-time PCR for the specific detection of parvalbumins, as the cases of the Atlantic and Pacific herrings (Clu h 1 and Clu pa 1) or the Pacific mackerel (Sco j 1) (Lee & Taylor, Citation2011; Rencova et al., Citation2013). Sun, Liang, Gao, Lin, and Deng (Citation2009) developed a real-time PCR assay for the specific detection of parvalbumin gene in fish, allowing a sensitivity of 5 pg. Concerning the detection and quantification of shellfish allergens, despite the preponderance of immunochemical methods, real-time PCR has only been applied to tropomyosin analysis in blue crab (Cal s 2) and tiger prawn (Pen m 1; LOD, 0.1–1 mg/kg; Eischeid et al., Citation2013).
Several factors should be considered when choosing a technique for the detection of allergens in foods, such as the availability of expensive equipment and experienced personnel, the time consumed per analysis, the cost of analysis, among others. For instance, MS platforms present reliable results, but require expensive equipment, specialised personnel and are not very suited for routine analyses. In the case of ELISA, the time per sample analysis is relatively short, the need for specialised personnel and cost are low/moderate, but the reliability of results can be compromised by cross-reactivity phenomena and food processing. DNA-based methods using quantitative technology such as real-time PCR require specialised personnel and equipment, moderate time of analysis, present high specificity and sensitivity, but can only provide indirect information regarding the detection of allergens in foods. In summary, all the methods present major advantages and also some drawbacks, so the choice of the method should be critically analysed according to the food matrix, type of processing and the available equipment (Costa et al., Citation2014). Whenever possible, the combination between protein- and DNA-based methods is highly recommended for confirmation and identification purposes.
Final remarks
Recent data suggest that there is a clear increase in the number of reported cases of allergy to seafood proteins, along with the growing consumption of fish and shellfish at a global scale The major allergens in fish and shellfish are parvalbumins and tropomyosins, respectively, with several occurrences as IgE-reactive proteins already reported, which have been currently characterised and publically made available in allergen databases. The establishment and development of novel methodologies for the detection and quantification of allergens in fish and shellfish are also of crucial importance for a better assessment and management of these proteins in processed foods. Protein-based assays such as ELISA, LFD and MS platforms are the most well-known used techniques for the detection/quantification of parvalbumins and tropomyosins in seafood products. Additionally, the use of DNA-based methods has already allowed the development of some commercial kits that detect different allergens by means of real-time PCR technology. Despite the available protein- and DNA-based methods, novel techniques allowing the detection and quantification of fish and shellfish in foods at trace amounts (with limits of detection as low as 1 mg/kg) are still much needed. Forthcoming advances should become available upon the development of testing/reference materials, the establishment of official methods for seafood allergen detection, as well as novel information regarding allergen threshold levels.
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References
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