Seafood hypersensitivity in mite sensitizedindividuals: is tropomyosin the only responsibleallergen?Manuel Boquete, MD, PhD*; Victor Iraola, PhD†; María Morales, PhD†; Helder Pinto, BSc†;Carballada, Francisco, MD*; Clara Carballás, MD*; and Jerónimo Carnés, PhD*

Background: Seafood allergy has been related to mite sensitization, mainly mediated by the muscle protein tropomyosin.Objectives: To determine the correlation between seafood hypersensitivity and mite sensitization (Dermatophagoides

pteronyssinus and Chortoglyphus arcuatus, a highly prevalent storage mite in Spain) and to investigate the implication oftropomyosin in cross-reactivity.

Methods: Patients from Northwest Spain were divided into 2 groups. The mite-seafood group contained 30 allergic miteindividuals with a clinical history of food hypersensitivity. The mite group contained 40 individuals with positive skin prick testresults to D pteronyssinus and C arcuatus but negative seafood test results. Specific IgE (sIgE) to whole mite and shrimp extracts,mite tropomyosin (rDer p 10), and shrimp tropomyosin (rPen a 1) were determined in each serum sample. Allergenic profileswere analyzed by immunoblot. Cross-reactivity studies were investigated by enzyme-linked immunosorbent assay and immu-noblot inhibition studies.

Results: In the mite-seafood group, 71% of patients had positive sIgE results to shrimp and 55% of them to shrimptropomyosin. A strong correlation was found between sIgE to shrimp tropomyosin and mite tropomyosin. Positive correlationwas observed between sIgE to shrimp tropomyosin and severity of symptoms. In the mite group, none of the 20% of patients withsIgE to shrimp tested positive to shrimp tropomyosin. In the immunoblot inhibition experiment, the shrimp extract was totallyinhibited by mite extract. These data suggest that primary sensitization is related to mite sensitization.

Conclusion: Tropomyosin does not seem to be the main allergen involved in mite-seafood sensitization in mite sensitizedindividuals. High levels of sIgE to tropomyosin seem to be related to severity of symptoms.

Ann Allergy Asthma Immunol. 2011;106:223–229.

INTRODUCTIONThe prevalence of seafood allergy is usually high whenconsumption of seafood plays a greater part in the diet of anobserved community. Previous studies have demonstratedthat the major allergen responsible for ingestion-related al-lergic reactions due to crustacean is the muscle protein tro-pomyosin.1 However, other characterized allergens, includinga 40-kDa arginine kinase,2 a 20-kDa protein identified as amyosin light chain,3 and recently a sarcoplasmatic calcium-binding protein, described in black tiger shrimp4 and whiteshrimp,5 can be responsible for allergic sensitization to sea-food.

On the other hand, seafood sensitization has also beenrelated to mite sensitization,5 mainly mediated by the highlyconserved muscle protein tropomyosin. This allergen hasbeen identified as the responsible allergen for cross-reactivitybetween seafood and mites. The molecular basis for this

cross-reactivity was reviewed by Ayuso et al,6 and it is basedon the amino acid sequence homology (81%) between mitetropomyosin (Der p 10) and shrimp tropomyosin (Pen a 1).This cross-reactivity has been proposed as the origin of thesensitization to shrimp tropomyosin after mite immunother-apy,7 although this de novo sensitization has been questionedin posterior studies.8,9 However, group 20 mite allergens(arginine-kinase, Der p 20), also present in decapod crusta-ceans, have recently been considered as new allergens in-volved in cross-reactivity between seafood and mites. Group20 mite allergens have been proposed as probable new pan-allergens,10 showing a 78% amino acid sequence homologywith Pen m 2. Recently, a new 20-kDa allergen present inshrimp and house dust mites (HDMs) has also been suggestedas responsible for cross-reactivity between crustaceans andHDMs.11 The authors speculate that this allergen could cor-respond to the sarcoplasmatic calcium-binding protein andmyosin light chain allergens.

Galicia is a region located in Northwest Spain. It is char-acterized by high exposure to mites, mainly the HDM Der-matophagoides pteronyssinus and the storage mite Chor-toglyphus arcuatus.12 The percentage of allergic patientssensitized to mites is very high, and it is usual to find patientsallergic to mites and with allergic symptoms, mainly oralallergy syndrome but also anaphylaxis, after ingestion of

Affiliations: * Allergy Service, Hospital General de Calde, Lugo, Spain;† R&D Department, Laboratorios LETI S.L. Tres Cantos, Madrid, Spain.

Disclosures: Authors have nothing to disclose.Received for publication July 9, 2010; Received in revised form October

18, 2010; Accepted for publication November 16, 2010.© 2011 American College of Allergy, Asthma & Immunology.

Published by Elsevier Inc. All rights reserved.doi:10.1016/j.anai.2010.11.014

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223

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seafood. The objectives of the study were to determine thecorrelation between sensitization to mites (D pteronyssinusand C arcuatus) and food hypersensitivity to crustaceans andto evaluate the cross-reactivity between shrimp and thesemites.

METHODS

Patient PopulationThe patient population consisted of 70 patients residing inNorthwest Spain (Lugo, Galicia) with allergic respiratorysymptoms (rhinoconjunctivitis and/or asthma) and with apositive skin prick test result to mites. All of them wereclinically studied about seafood allergy and skin prick testedwith biologically standardized (10 HEP/mL) D pteronyssinusand C arcuatus13 mite extracts (Laboratorios LETI, Madrid,Spain) and with 2 shrimp extracts (raw and boiled) at 5mg/mL. The patient population was divided into 2 groups.The mite-seafood group (MS group) consisted of 30 patients(13 females and 17 males; mean [SD] age, 31.4 [15.3] years).All of them had positive skin prick test results to D pteron-yssinus and C arcuatus and a clinical history of food hyper-sensitivity after seafood (shrimp) ingestion. According totheir clinical history and using the grading system proposedby Brown,14 12 patients (40%) reported oral allergy syndromeafter seafood ingestion, and 18 patients (60%) reported ana-phylactic reactions. Eight (26.7%) of them experienced amild reaction, 7 (23.3%) a moderate reaction, and 3 (10%) asevere reaction. The mite group (M group) consisted of 40control individuals (19 females and 21 males; mean [SD] age,31.6 [15.3] years). These patients had positive skin prick testresults to D pteronyssinus and C arcuatus but negative resultsto shrimp extract, and they did not show allergic symptomsafter seafood ingestion.

Wheal sizes were measured by papulometry using thesoftware PC Draft connected to Wacom Graphic tablet andGraphire 2 pencil (Wacon Company Ltd, Tokyo, Japan). Theresults were expressed in millimeters squared and were con-sidered positive when the reaction was 7 mm2 or greater.

A total of 40 serum samples from the M group and 28 fromthe MS group were obtained and frozen at –20°C. A pool ofsera was prepared mixing 16 serum samples from the MSgroup individuals with detectable (�0.35 kU/L) specific IgE(sIgE) against shrimp and C arcuatus. The serum pool wasaliquoted and stored at –20°C.

This study was approved by the ethics committee of thehospital, and written informed consent was obtained from allthe participants.

Extract PreparationShrimp (Penaeus longirostris, one of the most commercial-ized shrimp species in Spain) were bought at a local marketand used to prepare raw and boiled extracts. Briefly, wholeraw shrimp or shrimp previously boiled in 0.01M phosphate-buffered saline (PBS) for 15 minutes were individuallyplaced in a blender for 3 minutes in 0.01M PBS until the

material was totally homogenized. Subsequently, both raw r

224

nd boiled extracts were kept under continuous magnetictirring for 4 hours at 4°C, centrifuged, dialyzed overnightgainst bidistilled water in 3.5-kDa membranes, sterile fil-ered, and freeze-dried. The protein content was measured byhe Lowry-Biuret method (BioRad Laboratories, Hercules,alifornia).

IgE DeterminationIgE to D pteronyssinus, nDer p 1, rDer p 2, rDer p 10 (miteropomyosin), shrimp, and recombinant shrimp tropomyosinrPen a 1) were measured using the UniCAP system (Phadia,ppsala, Sweden). Specific IgE to C arcuatus was deter-ined using the manufactured extract with the UniCAP sys-

em. Briefly, C arcuatus extract was biotin labeled (Rocheiagnostics, Indianapolis, Indiana) and incubated with

treptavidin ImmunoCAPs. After washing, sIgE was mea-ured following the manufacturer’s instructions (Uni-APs100; Phadia).

DS and Immunoblothe protein profile of raw shrimp and C arcuatus extractsere determined by sodium dodecyl sulfate–polyacrylamideel electrophoresis (SDS-PAGE). Shrimp and C arcuatusxtracts were run in SDS-PAGE gels with 2.67% C, 15% Tcrylamide as described by Laemmli.15 A total of 100 �g ofrotein was loaded onto the gels and Coomassie stained.Allergenic profiles of shrimp and C arcuatus extracts were

tudied by immunoblot, using individual serum samples.riefly, after electrophoresis, gel was electrotransferred to a-immobilon membrane (Millipore, Bedford, Massachusetts)nd dried at room temperature. Thereafter, membranes werencubated overnight with individual serum samples (1:2 di-ution) and developed using antihuman IgE (Ingenasa, Ma-rid, Spain) labeled with horseradish peroxidase. The reac-ion was visualized by chemiluminescence.

LISA Inhibitionlastic microtiter plates (Immulon IV; Dynex Technologies,hantilly, Virginia) were coated with C arcuatus extract (10g/mL of protein) and incubated overnight. Several dilutionsere made from raw shrimp and C arcuatus extract. Eachilution was incubated with the serum pool for 2 hours atoom temperature. Afterward, 100 �L was transferred to thehrimp-coated plate and incubated overnight. After washing,00 �L of peroxidase-labeled anti-human IgE (Ingenasa) wasdded and allowed to stand for 30 minutes at room temper-ture. After washing, the plates were developed for 30 min-tes and stopped with 1N sulfuric acid.

mmunoblot Inhibition Experimentsmmunoblot inhibition experiments were performed usingaw shrimp and C arcuatus extracts in solid phase. A total of00 �g of proteins of each extract was transferred to aitrocellulose membrane following the procedure previouslyescribed. The serum pool was incubated beforehand for 2ours with C arcuatus (in case of shrimp in solid phase) or

aw shrimp extract (500 �g/mL) (in case of C arcuatus in

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solid phase) and then added to the membranes and incubatedovernight. After washing, membranes were incubated withanti-human IgE peroxidase (Ingenasa), developed with lumi-nol solution (Immun-Star HRP Chemiluminescent Kit; Bio-Rad), and detected using chemiluminescence (ChemiDocXRS; BioRad).

Statistical AnalysisThe statistical analysis consisted of descriptive and nonpara-metric statistics. Wheal sizes and sIgE were compared be-tween both groups of patients using the Mann-Whitney test.Differences in each group of patients were calculated usingthe Mann-Whitney test, Wilcoxon signed rank test, and mul-tiple comparisons for analysis of variance on ranks (Dunntest). Proportions were analyzed using contingency tables,and the Fisher exact test was conducted. P � .05 was con-sidered statistically significant.

RESULTS

Comparison Between Groups of PatientsSkin prick tests. Table 1 lists the percentage of individuals

with positive skin prick test results and the wheal sizesobtained with the different extracts. Wheal sizes with Carcuatus were higher in the MS group than in the M group;the opposite happened with the D pteronyssinus extract. Sta-tistically significant differences were not observed in anygroup.

In the MS group, wheal sizes induced by mites were largerthan those induced by shrimp. After comparing shrimp ex-tracts, raw extract elicited larger wheals than those inducedby boiled extracts but without significant difference. Therewas a significant correlation between wheal size induced byraw and boiled shrimp (P � .001).

In this group there was an increase in the percentage ofpatients with positive skin prick test results to shrimp with theseverity of symptoms; 75% of the individuals with mild symp-toms, 85.7% of those with moderate symptoms, and 100% withsevere symptoms had positive skin prick test results to shrimpextract.

Specific IgE. Table 2 gives the mean level and percentageof patients with detectable sIgE (�0.35 kU/L) in both groups.Levels of sIgE to D pteronyssinus, Der p 1, Der p 2, and Carcuatus were clearly higher in the M group compared withthe MS group, with statistically significant differences in thecase of D pteronyssinus (P � .005), Der p 1 (P � .008), and

Table 1. Patients With Positive Skin Prick Test Results to Mite and S

Extract

Patient population (M

Mite and seafoodpositive, %

Wh

Raw shrimp extract 80.0Mite extracts

Dermatophagoides pteronyssinus 100

Chortoglyphus arcuatus 100 101.3

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er p 2 (P � .007). In both group of patients, sIgE levels toer p 2 were statistically significant higher than to Der p 1

MS group: P � .003; M group: P � .002). In the M group,small percentage of patients had detectable sIgE to shrimp,ith a mean value statistically lower (P � .001) than thatbtained in the MS group. Levels of sIgE to shrimp and miteropomyosin, Der p 10, were only detected in the group of

S patients. sIgE levels to Der p 10 were lower than thosebtained in Der p 1 and Der p 2, although the difference wasnly significant with Der p 2 (P � .02). However, it isoteworthy that 13% of the patients have sIgE to Der p 10 butot to Der p 1 or Der p 2. In the MS group, 55% of theatients with sIgE to shrimp had sIgE to shrimp tropomyosinoo. A high correlation between shrimp and mite tropomyosinP � .001) was detected. Regarding differences in sIgE levelso D pteronyssinus and C arcuatus between patients with orithout sIgE to rPen a 1, no significant differences werebserved (P � .05).Figure 1 shows the sIgE levels to shrimp and shrimp

ropomyosin in the MS patients, sorted according to severityf symptoms after ingestion of shrimp. There was a signifi-ant difference among sIgE to rPen a 1 in the different groupsP � .03), with the level of sIgE to rPen a 1 being statisticallyigher (P � .05) in patients with severe reactions than inhose with oral allergy syndrome or mild reactions aftereafood ingestion. No significant differences were observedith sIgE to shrimp or mites.

ross-reactivity Between C arcuatus and ShrimpAntigenic profiles of C arcuatus and shrimp extracts. Pro-

ein profile of C arcuatus and shrimp extracts, analyzed byDS, and matching between both extracts evidenced theresence of common proteins with the same molecular weightn both extracts (Fig 2). The individual immunoblots (Fig 3)learly showed a higher and more diverse recognition patterno C arcuatus than to shrimp extract.

ELISA inhibition experiments. The results of the ELISAnhibition experiments showed that the amount necessary toeach the 50% inhibition point using C arcuatus extract in theolid phase was 0.237 �g for C arcuatus, whereas the shrimpxtract did not inhibited mite extract, suggesting a lack ofross-reactivity between C arcuatus and shrimp, using theerum samples of patients sensitized to both.

Immunoblot inhibition experiments. Figure 4 shows themmunoblot inhibition experiments using C arcuatus or

Extracts

� 30) Control population (M) (n � 40)

ze, meanmm2

Mite positive and seafoodnegative, %

Wheal size, mean(SD), mm2

(51.6)

(103.3) 100 143.5 (90.5)

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(82.9) 100 69.5 (42.5)

225

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shrimp extract in solid phase. Only bands with molecularweights lower than 21 kDa were inhibited using C arcuatusin solid phase. However, inhibition experiment between Carcuatus and shrimp, using shrimp extract in the solid phase,showed a practically total inhibition. Only a weak recognitionof a 25 kDa was detected.

DISCUSSIONThe relationship between shrimp and HDM allergy is wellknown. Tropomyosin is the usual suspect protein in mite-crustacean cross-reactivity because it is present in bothgroups with a high sequence homology.6 However, althoughtropomyosin is an important allergen in crustaceans, it isconsidered as a minor allergen in HDMs and storage mites.16

The existence of mite sensitized individuals who do notrecognize mite tropomyosin but with adverse reactions aftercrustacean ingestion suggests the existence of other respon-sible allergens. Besides, there is little information about the

Figure 1. Box plot of specific IgE (ImmunoCAP) against shrimp, tropom

Table 2. Patients With Detectable sIgE (�0.35 kU/L) and sIgE Levels

Patient population (MS)

Mite and seafoodpositive, %

s

Shrimp 71.4Shrimp tropomyosin (rPen a 1) 39.3Dermatophagoides

pteronyssinus89.3

Chortoglyphus arcuatus 71.4Mite tropomyosin (Der p 10) 32a

Der p 1 39.1a

Der p 2 47.8a

Abbreviation: sIgE, specific IgE.a Performed in 25 patients.

patients in the mite-seafood group sorted according to severity of symptoms after i

226

ole of storage mites in these symptoms, although the lack ofross-reactivity between shrimp and storage mites has beenescribed.17

In the present study, we compared the different sensitiza-ion profiles between 2 groups of individuals sensitized to theost abundant mites in Northwest Spain, D pteronyssinus

nd C arcuatus. Patients from one of the groups experiencedllergic symptoms after seafood ingestion. By contrast, thether group did not experience adverse reactions after crus-acean ingestion. Besides, cross-reactivity between C arcu-tus and shrimp, using serum of patients sensitized and withIgE to both of them, was also determined. It is worthentioning that, although no C arcuatus allergens have been

oted by the International Union of Immunological Societies,he C arcuatus tropomyosin has been cloned, and it presentshigh sequence homology (94%) with Der p 10.18

More than 70% of patients with symptoms after crustaceanngestion had sIgE to shrimp, and 55% of them had sIgE to

Pen a 1), Dermatophagoides pteronyssinus, and Chortoglyphus arcuatus in

8) Control population (M) (n � 40)

vel, Mite positive and seafoodnegative, %

sIgE level, mean(SD), kU/L

2.4) 20 0.2 (0.4)0.3) 01.8) 100 32.2 (27.8)

.5) 92.5 6.2 (6.7)

.9) 02.1) 77.5 11.8 (17)0.2) 85 18.7 (20.7)

yosin (r

(n � 2

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5 (13.9 (1

20.9 (3

3.8 (41.6 (35.6 (1

10.9 (2

ngestion of shrimp.

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pshtti

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tropomyosin. This percentage is lower than those described inprevious published articles,19,20 in which 70% to 80% ofpatients with shrimp allergy had sIgE to shrimp tropomyosin.In the control group, a small percentage of individuals haddetectable sIgE to shrimp, although these values were low.No sIgE to raw tropomyosin was observed in this group.

According to our results, sIgE to tropomyosin is a markerof the severity of allergic symptoms after seafood ingestionbecause the level of sIgE to rPen a 1 was higher in patientswith severe reactions than in those with oral allergy syndromeor mild reactions. Regarding the sIgE levels to mites, statis-tically significant differences were observed between the 2groups, with higher levels of sIgE to D pteronyssinus, Der p1, and Der p 2 in control patients (M group) and with sIgE toDer p 10 detected only in patients (30%) with symptoms aftercrustacean ingestion. A noteworthy finding is that 13% ofpatients had sIgE to mite tropomyosin but not to Der p 1/Derp 2. Besides, differences in sIgE to the different mite aller-gens into each group were found, with dominance of sIgE toDer p 2. Similar results were observed in mite allergic pa-tients residing in Northwest Spain.21 These results seem to

Figure 2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis andC arcuatus; lane 3, raw shrimp.

confirm the importance of sensitization to tropomyosin in c

VOLUME 106, MARCH, 2011

atients allergic to mites and crustaceans, related to theeverity of symptoms. However, it is also remarkable that aigh percentage of mite sensitized patients who have symp-oms after shellfish ingestion did not show detectable sIgE toropomyosin and they did not recognize this allergen in themmunoblot.

Cross-reactivity was investigated by ELISA and immuno-lot inhibition experiments. These studies suggest a lack ofross-reactivity between C arcuatus and shrimp, when a poolf serum from patients sensitized to both allergens were used.his finding has been observed in previous studies,17 in whichespite the fact that tropomyosin is found to be a commonllergen in mites, there is no cross-reactivity between mitesincluding C arcuatus) and shrimp. A possible explanation iselated to the different tropomyosin concentrations in mitend shrimp extract, with tropomyosin being the dominantrotein in shrimp extract because crustaceans are almostntirely muscle, whereas mites have little muscle. In addition,multivariate statistical analysis of IgE antibody serum con-

entrations of a high number of sensitized individuals to 89llergen extracts showed the reactivity to crustaceans was

ng of Chortoglyphus arcuatus and shrimp extract. Lane 1, standard; lane 2,

matchi

lustered with cockroach but not with mites.22

227

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. A, Ra

In our study, in a population sensitized and exposed to highlevels of mites and with a clinical history of food hypersen-sitivity after crustacean ingestion, the results (higher whealsize and sIgE levels to mites than those to shrimp extracts)suggest that sensitization to mites is the primary cause of

Figure 3. Immunoblot using individual patient’s serum

Figure 4. Immunoblot inhibition. Lane 1, Solid-phase Chortoglyphusarcuatus (not inhibited); lane 2, solid-phase raw shrimp extract (not inhib-ited); lane 3, solid-phase C arcuatus (inhibited with raw shrimp extract); lane

s4, solid-phase raw shrimp extract (inhibited with C arcuatus).

228

llergy, with food hypersensitivity being a consequence ofross-reactivity. Similar findings have been described inther places with high exposure to mites, such us Martin-que.23 This fact is confirmed by the allergenic profile of ouratients because more than half of the patients had a high andiverse recognition pattern in the mite extract, whereas onlyfew patients recognize shrimp proteins. Besides, the immu-oblot inhibition experiment showed an almost completenhibition of the shrimp extract by the mite, whereas onlyite proteins lower than 21 kDa were inhibited by the crus-

acean.However, in the case of the patients sensitized to tropo-yosin, the primary sensitization to this protein by mites is

ot so clear. The theory that the sensitization to shrimpropomyosin could be due to respiratory allergens, such asites and/or cockroaches, has been confirmed in non–crus-

acean-eating patients, such us orthodox Jews.24 However, inur case the primary sensitization to tropomyosin could hap-en through ingestion of seafood better than through respi-atory sensitization to mites. The higher sIgE levels to shrimpropomyosin than to the mite tropomyosin, detected in someatients, seems to substantiate this supposition.In summary, and according to our results, tropomyosin

oes not seem to be the major allergen involved in mite-eafood sensitization in mite sensitized individuals with mildymptoms, mainly oral allergy syndrome, after crustaceanngestion. The implication of other allergens should be fur-her analyzed in detail. However, when mite allergic patientsave severe reactions to crustacean intake, tropomyosin cane considered as an important allergen. Regarding the role ofhe prevalent storage mite C arcuatus in cross-reactivity with

w shrimp extract; B, Chortoglyphus arcuatus extract.

hrimp, we consider that their importance is clearly lower

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1

1

1

1

1

1

2

2

2

2

2

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than those observed with D pteronyssinus. Further studies areneeded to clarify the implication of other allergens in theseafood-mite syndrome.

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1. Iraola V, Boquete M, Pinto H, Carballada F, Carballás C, Carnés J.Pattern of sensitization to major allergens Der p 1 and Der p 2 inmite-sensitized individuals from Galicia, Spain. J Investig Allergol ClinImmunol. 2010;20:270–272.

2. Soeria-Atmadja D, Onell A, Kober A, et al. Multivariate statisticalanalysis of large-scale IgE antibody measurements reveals allergenextract relationships in sensitized individuals. J Allergy Clin Immunol.2007;120:1433–1440.

3. Purohit A, Shao J, Degreef JM, et al. Role of tropomyosin as a cross-reacting allergen in sensitization to cockroach in patients from Martin-ique (French Caribbean island) with a respiratory allergy to mite and afood allergy to crab and shrimp. Eur Ann Allergy Clin Immunol. 2007;39:85–88.

4. Fernandes J, Reshef A, Patton L, et al. Immunoglobulin E antibodyreactivity to the major shrimp allergen, tropomyosin, in unexposedorthodox Jews. Clin Exp Allergy. 2003;33:956–961.

equests for reprints should be addressed to:erónimo Carnés, PhDaboratorios LETI S.L. Calle del Sol. 28760 Tres Cantosadrid, Spain

toglyphus arcuatus. Ann Allergy Asthma Immunol. 2006;9:203–208. E-mail: jcarnes@leti.com

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