Allergens and molecular diagnostics of shellfish allergy

Shellfish belongs to “The Big 8” food groups causing allergy, which often does not outgrow during childhood. Shellfish is one of the main food allergens in adults and constitutes a diverse group of species subdivided into crustaceans and mollusks, which seem to include similar but also different allergens. Several pan-allergens are characterized in detail, including tropomyosin and arginine kinase, responsible for clinical cross-reactivity with other invertebrate allergen sources, embracing mites, insects, and parasites. Currently, at least seven different shellfish allergens have been identified, mostly from crustaceans. However, only three recombinant allergens are available for IgE-based routine diagnostic, including tropomyosin, arginine kinase, and sarcoplasmic Ca2+-binding protein. Other allergens include myosin light chain, troponin C, triosephosphate isomerase, and actin. This review summarizes the current advances on the molecular characterization of shellfish allergens, clinical cross-reactivity, and current diagnostic approaches for the management of this life-threatening disease.

ular, the oral allergy syndrome (OAS) seems to be very o en experienced by crustacean allergic subjects. Shrimp has also been implicated in food-dependent exercise-induced anaphylaxis [4]. Currently, 2 % of the general world population is af-% of the general world population is af-% of the general world population is af fected by shell sh allergy, with much higher rates in countries with high seafood consumption. Unlike many other food allergies, most shell sh allergy persists for life in the a ected individual.

Classi cation of shell sh groups
Patients with allergy to shell sh may fail to identify the o ending seafood species, o en as a result of confusion regarding the di erent common names used to describe diverse seafood. e two invertebrate phyla of arthropods and mollusks are generally referred to as "shell sh" (Fig. 1).
Crustaceans are, perhaps surprisingly, classi ed as arthropods together with spiders and insects.
is might provide an explanation for the observed molecular and clinical cross-reactivity discussed in detail below. e group of mollusks is a large and diverse group, subdivided into the classes bivalve, gastropod, and cephalopod, including several important seafood groups including mussels, oysters, abalone, snails, and squid (calamari).

Prevalence of shell sh allergy
e prevalence of allergic reactions to seafood is usually higher when the consumption plays a greater part in the diet of the observed community. It is generally considered that crustaceans and mollusks are among the foods that most commonly provoke severe anaphylaxis [6]. A recent study established surprisingly that seafood allergies are a signi cant health concern a ecting approximately 6.5 million people in the United States (US) -more than twice as common as peanut allergy. e telephone survey among 14,948 individuals reported 5.9% with shellsh allergy, and seafood allergy was almost 5-times more common among adults compared to children [7]. In France, in a study by Andre and co-workers among 580 patients with adverse reactions to food, 34 % were identi ed having speci c IgE to crab [8].
A study from South Africa including 105 individuals with perceived adverse reactions to seafood conrmed sensitization to shrimps and rock lobster in almost 50 % [1,9].
While seafood allergy is common in Western countries such as Europe, the US, and Australia, it seems that in Asian countries allergic reactions to shell sh are of greater importance among adults and children [10,11,12]. is clearly supports the   view that the likelihood of becoming sensitized to shell sh seems to correlate with geographical eating habits and is most likely underreported in many Asian populations.
Not only ingestion of shell sh can cause sensitization, but also exposure during processing in factories and domestic environment. ere seems to be a strong correlation between high concentration of air-borne allergens and increased allergic sensitization [13,14,15,16].

Structure and biological functions of shell sh allergens
Over the past 20 years, several shell sh allergens, particularly in crustaceans have been identi ed and sequenced. Currently, 34 allergens have been identi ed and characterized in detail from various crustacean and mollusk species and registered in the International Union of Immunological Societies (IUIS) Allergen Database (Tab. 1) (www. allergen.org) [17]. e biochemical characteristics of shell sh allergenic proteins are typically of low molecular weight, high water solubility, high heat stability, and an acidic isoelectric point. Almost all of the known characterized allergens are found in the edible portions of various shell sh species. However, some protease-based allergens, (non-IgE-mediated), are present in the gastrointestinal regions of the di erent shell sh species [18]. e allergen family speci c properties of shell sh allergens are described below and summarized in Tab. 1 and Tab. 2.
Tropomyosin Pen m 1 Tropomyosin (TM) is the major allergenic protein across all edible crustacean and mollusk species. More than 60 % of shell sh allergic patients are sensitized and react to TM, o en leading to severe systemic reactions. Tropomyosin-speci c IgE is frequently used to predict clinical outcomes of shrimp allergy with a positive predictive value of 0.72 [19,20]. is allergen is an alpha-helical coiled-coil dimeric protein that binds along the length of actin and regulates the cooperation of troponin and myosin, thus controlling the contraction of muscle bers [21]. e primary structure is highly conserved across various invertebrate species. is seems to be the main reason for high IgE-mediated allergenic cross-reactivity across various shell sh species as described below in detail. Interestingly, even though crustacean and mollusk TMs are allergenic, they share only very low amino acid sequence identities of 55 to 70 %.
Allergenic TMs have generally molecular weights of between 33 and 38 kDa and are highly stable to heat-treatment, capable of retaining allergenicity even a er cooking and high-pressure processing. According to the Allfam database (www.meduniwien.ac.at/allfam), the TM family is the largest "food" allergen family in animal sources, consisting of currently 47 identi ed TMs, mostly from crustacean species [17].

Arginine kinase Pen m 2
Arginine kinase (AK) has been identi ed in over six crustacean and one mollusk species. IgE sensitization to the 40-42 kDa AK has been demonstrated in 21-50 % of adults and 67 % children [22,23]. Although heat labile, IgE binding has been demonstrated to AK in heat-treated shrimps, which may be due to remaining intact IgE epitopes on aggregated AK [22,24]. Interestingly, crustacean AK along with TM has also been implicated in inhalational exposure and sensitization among crab processing workers [25]. Crustacean AK has been demonstrated to cross-react to ingested insect AK as well as being implicated in seafood-mite cross-reactivity [26,27].

Myosin light chain Pen m 3
e EF hand domain superfamily is the second largest group of all allergens, a er pro lins, which encompasses both food and inhalant allergens from animal and plant sources. ree classes of shell sh allergens are EF hand domain proteins, which include myosin light chain (MLC), sarcoplasmic calcium binding proteins, and troponin.
MLC is mainly found in smooth muscles in complex with myosin heavy chain motor domains. Myo-

Penaeus indicus
Indian white prawn

Homarus americanus
American lobster Hom a 1 Hom a 3 Hom a 6 Panulirus stimpsonii sin light chains have a molecular weight between 17 and 20 kDa, are well characterized in four crustacean species and seem to be heat stable. Currently, there is a lack of data on immunological cross-reactivity of MLC among crustaceans, mollusks, or other invertebrate species.

Sarcoplasmic calcium binding protein Pen m 4
Sarcoplasmic calcium binding proteins (SCBPs) are also members of the EF hand calcium binding protein family incorporating the helix-loop-helix motif in the primary amino acid sequence. It has a molecular weight of approximately 20 kDa and an isoelectric point of 5, and can elicit IgE binding even a er heat treatment [22]. Recent studies have highlighted the relevance of SCBP as a shell sh allergen. Ayuso et al. demonstrated IgE recognition in 85 % of shrimp allergic children, which is much higher compared to tropomyosin [28]. More importantly, it has been shown that speci c IgE to SCBP, in addition to that of TM, is associated with clinical reactivity to shrimps [20].

Troponin C Cra c 6
Troponin C (TnC) has been characterized in shrimps, but also as important cockroach allergen (Bla g 6 and Per a 6). Similar to SCBP and MLC, TnC is an EF hand calcium binding protein. Troponin C is approximately 20 kDa in size and its possible heat stability is not fully understood. e IgE binding frequency to TnC is with 15 % lower as reactivity to TM, AK, or SCBP.

Triosephosphate isomerase Cra c 8
Triosephosphate isomerase (TIM) has been characterized in shrimps (Cra c 8), cray sh (Arc s 8), and cockroach (Bla g TPI). It has an approximate molecular weight of 28 kDa and is probably heat sensitive [29]. e clinical and immunological cross-reactivity of TIM among various invertebrate species are not well understood and amino acid sequences have not been performed.

Clinical and immunological cross-reactivity
True sensitization to shell sh speci c allergens can be hampered due the highly cross-reactive nature of some allergenic proteins. e most well known pan-allergen is tropomyosin, being the major cause for reported clinical cross-reactivity among and between crustaceans and mollusks, but also other invertebrates, including mites, cockroaches, and parasites (Fig. 2). It is known that tropomyosin has mainly linear IgE epitopes and is of great importance in determining the degree of cross-reactivity between di erent shell sh species. Tropomyosin is highly conserved among various crustacean species such as prawn, crabs, and lobsters with amino acid identities reaching 95-100 %. erefore, IgE crossreactivity is very frequent among crustacean species [30,31,32,33,34]. Within the mollusk group, hypersensitivity cross-reaction is o en seen in allergic individuals, as determined for ten di erent species of cephalopods [35]. Similar results were shown for four species of gastropods (disc abalone, turban shell, whelk, and Middendorf's buccinum) and seven species of bivalves (bloody cockle, Japanese oyster, Japanese cockle, surf clam, horse clam, razor clam, and short neck clam) [36].
Increasingly important seems to be IgE cross-sensitization between tropomyosin from shell sh and other important allergenic invertebrates, including dust-mites and cockroaches (Fig. 2). It was demonstrated that IgE against mite tropomyosin (Der p 10) reacted very strongly to shrimp tropomyosin, although tropomyosin is present in very low concentrations in house dust mites [37]. More interestingly, reactivity to shrimp has been demonstrated in subjects with house dust-mite allergy, who have never been exposed to shrimps due to religious eating habits [38].

Potential advantages of componentresolved diagnosis in shell sh allergy
Applying single allergenic molecules from shell sh for allergen-speci c IgE detection could potentially modify 1. test sensitivity (improving the limit of quantitation to shell sh allergens of rare abundance or low stability) and/or modify 2. analytical speci city, particularly if speci c IgE is detectable to a) risk associated molecules (being more likely responsible for severe reactions and/or more speci c for children or adults), b) indicators of cross-reactivity (involved in broad serological cross-reactions between different shell sh species), c) markers of primary species-and/or familyspeci c sensitizations (facilitating the identication of unique allergic sensitizations to certain shell sh species or families). e listed advantages of component-resolved diagnosis (CRD) require some allergen-related knowledge about abundance of single allergens in the shell sh body (and resulting extracts), -location of the allergen in the organism (edible or non-edible parts), water solubility (for proper extraction), -stability and behavior to thermal and gastric degradation, -frequency of sensitization to the single allergen in question, -degree of inter-species or inter-family related cross-reactivity, -risk to elicit severe allergic reactions. Speci c IgE to TM, thanks to its high abundance and stability, is picked up reasonable easily using heated protein extracts from probably most shellsh species. us, there is no particular need to further increase test sensitivity. However, increased ana lytical speci city of TM in molecular-based serological tests will help to identify patients at risk for severe allergic reactions and, in addition, indicate broad cross-reactivity to TM from other shellsh species and perhaps insects and mites. Testing IgE to more than one TM is probably providing more information about cross-reactivity between crustaceans and mollusks.
Similar assumptions are related to the other described shell sh allergens (see above), i. e., AK, MLC, SCBP, TnC, TIM: being part of the edible part of shell sh, with basic functions in musclebers or general energy metabolism, they are presumably also highly conserved, showing variable degrees of cross-reactivity, which has not been studied yet. Increasing test sensitivity through the use of single molecules might be useful in less stable allergens (i. e., AK, TIM), but not necessarily for more robust proteins (i. e., MLC, SCBP). Increased analytical speci city can assist uncovering associated risks, i. e., in case of IgE to SCBP [20]. However, none of these candidates might serve as a single marker for species-speci c sensitization due to variable degrees of IgE-related cross-reactivity, which still needs to be addressed. Recent advances in PCR based allergen-speci c IgE quanti cation have further improved the sensitivity and speci city to single allergens, using serum from a nger-prick, which is of particular advantage for infant allergy testing [39].
In conclusion, no species-speci c allergens have been identi ed so far, making it di cult to precisely diagnose allergy to a speci c crustacean or mollusk species with the use of allergen molecules [3,40]. If more of the already identi ed and additional allergens are available for diagnostics, it might be helpful to test one per protein family, ensuring maximum test sensitivity and enhanced molecular speci city, particularly if TM is not the major allergen.
is does, however, not solve the question of potential clinical cross-reactions to closely related shellsh species: only anamnestic data or oral challenges can indicate or rule out clinically relevant allergic reactions to certain shell sh species.

Diagnostics separating IgE-mediated allergy from other reactions
Serum based IgE quanti cation tests are available for a wide variety of crustacean and mollusk species as well as for cross-reactive invertebrate species such as dust-mites and cockroaches. IgE quantication tests for single component allergens are currently only available for shrimp tropomyosin (rPen a 1). However, some additional shell sh allergens are available in multiplex (microarray) format for prawn tropomyosin (nPen m 1), arginine kinase (nPen m 2), and sarcoplasmic calcium binding protein (rPen m 4).
Approximately 60 % of patients with clinical allergy to crustacean demonstrate speci c IgE binding to tropomyosin. It has been suggested that IgE reactivity to tropomyosin is a better predictor of shrimp allergy as compared to skin prick testing (SPT) or IgE to whole shrimp extract [19,23]. However, also sarcoplasmic calcium-binding protein (Pen m 4) reactivity has been associated with clinical reactivity to shrimp. e combination of reactivity to both allergens might increase the sensitivity to detect clinically allergic patients, but has still to be con rmed.
e consumption of seafood is very di erent from most other food allergen sources. It can trigger clinical adverse symptoms, although non-allergic in origin, being similar in clinical presentation to true IgE-mediated allergic reactions. ese substances are found in seafood much more frequently as compared to any other food source. An atypical clinical history or an inconsistent history always suggests a non-atopic etiology, such as contamination with marine bio-toxins, parasites, bacteria, and viruses [41,42]. Because of the similarity in clinical reactions of a ected individuals, it is essential to di erentiate adverse reactions from true shell sh allergy and understand the molecular nature of the o ending allergens for improved component-resolved diagnosis.
Food challenge or double blind placebo controlled food challenge (DBPCFC) can be performed to con rm clinical reactivity to crustacean and mollusk species. However, such provocation tests are not performed routinely because of increased risk and costs, and are only performed for investigating individual cases.

Outlook for future diagnostic options
Most of the clinical studies on cross-reactivity have been conducted using tropomyosin as the major pan-allergen. However, other shell sh allergens may play a role in immunological cross-sensitization. A recent study has shown that allergens other than tropomyosin, such as arginine kinase, might also be responsible for cross-reactivity between shell sh and inhalant invertebrate allergen sources [27,43]. In addition, hemocyanin has been demonstrated to be cross-reactive and is also a known cockroach allergen [44,45].
However, an in-depth investigation into the conservation or relevance of speci c IgE epitopes between pan-allergens from crustaceans and mollusks and clinical cross-reactivity to mites and cockroaches has not been conducted or con rmed using a larger number of shell sh allergic patients.

Suggestions for present clinical practice
Diagnosis of shell sh allergy is based on clinical history, -sensitization tests (allergen-speci c IgE tests; skin tests), and oral challenge test, if needed. In case of severe allergic reaction, allergen-speci c IgE should precede any in vivo tests, i. e., SPT, to avoid any risks for the shell sh allergic patient. IgE diagnostics should include total IgE (for improved interpretation of the quantitative allergen-speci c IgE values), -allergen-speci c IgE preferably to the reactioneliciting (or biologically closely related) shell sh species, -allergen-speci c IgE to Pen a 1, the currently only available TM for singleplex testing from brown shrimp (Penaeus aztecus). A step-by-step guide could be as follows: a) If extract-and TM-speci c IgE results are positive with quantitative IgE-levels being higher to TM than to the whole extract, immunodominant sensitization to shell sh TM is likely, and broad (serological) cross-reactivity to other shell sh species is to be expected. During interpretation of the test, concordance between recorded symptoms and the identi ed shell sh species should be checked. Only in case of corresponding symptoms and a positive sensitization test, clinically relevant allergy has successfully been demonstrated. b) If only the extract-speci c IgE, but not the TM-speci c IgE is positive, sensitization to TM is unlikely, but other shell sh allergens might be involved. c) If both IgE-tests (shell sh extract-and TM-speci c IgE) turn out to be negative, it is mandatory to perform a skin test, i. e., SPT with a commercial shell sh extract and/or a (titrated) SPT with native material (i. e., prick-prick-test with fresh shell sh species, if possible raw and cooked). d) In case of a clearly positive SPT result an immediate-type sensitization is likely, particularly if healthy control individuals do not react to the applied skin test material. e) In case of clearly negative skin test results, IgE-mediated sensitization to the tested shell sh species becomes very unlikely and di erential dia gnoses other than IgE-mediated allergic reactions to shell sh should be considered. f) Additional testing with other shell sh species has limited value for subsequent consulting of the patient: in case of positive skin or IgE test results, serological cross-reactivity has been demonstrated, which does not always translate into clinical cross-reactivity. However, in case of a clearly negative skin and/or IgE response to related or biologically more distant shell sh speci es (serological) cross-reactivity and subsequent clinical cross-reactivity becomes unlikely. g) In case of doubt or mismatch between case history and diagnostic results, carefully titrated oral challenge tests with the suspected shell sh species might solve the discrepancies. However, due to the risk for the patient in case of previous severe allergic reactions and limited specialized centers, they are not frequently performed. A negative provocation test, if previous sensitizations tests turned out negative, is usually safe and an appropriate way to rule out a present food allergy to shell sh. In general, patients with proven shell sh allergy should avoid a broad range of related shell sh species (crustacean or mollusk), unless they have already tolerated other (presumably biologically more distant) shell sh species. is rather cautious approach takes into account that allergic subjects are not necessarily familiar with huge variety of pre sent shell sh species, their biological relationship and the composition in mixed seafood dishes, particularly from non-self prepared meals.
Due to the o en long-lasting nature of IgE-mediated allergies to shell sh species patients with proven allergic reactions should avoid shell sh permanently, unless subsequent controlled challenges have ruled out a still present clinical reactivity.