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Keywords:

  • allergen;
  • cross-reactivity;
  • double-blind food challenge;
  • dust mites;
  • food allergy;
  • recombinant;
  • shrimp;
  • tropomyosin

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Authors’ contributions
  8. Conflict of interest
  9. References

To cite this article: Gámez C, Sánchez-García S, Ibáñez MD, López R, Aguado E, López E, Sastre B, Sastre J, del Pozo V. Tropomyosin IgE-positive results are a good predictor of shrimp allergy. Allergy 2011; 66: 1375–1383.

Abstract

Background:  Shrimp is a common cause of food allergy. Our aims were to determine the value of IgE antibodies in the diagnosis of shrimp allergy and to study red shrimp (Solenocera melantho) tropomyosin both as a new allergen and as a cross-reactive IgE-binding protein.

Methods:  We have studied 45 subjects. Skin prick test (SPT) was carried out in all subjects, and specific IgE (sIgE) to shrimp, recombinant and natural shrimp tropomyosins rPen a 1 and nPen m 1, recombinant Der p 10, and Dermatophagoides pteronyssinus was assessed by fluoroimmunoassay and/or immunoblotting. Double-blind, placebo-controlled food challenges were carried out to confirm diagnosis of shrimp allergy. Also, in vitro inhibition tests were performed to evaluate cross-reactivity.

Results:  Shrimp allergy was confirmed in 18 shrimp-allergic patients. Skin prick test and IgE antibodies to shrimp were positive in all shrimp-allergic patients; sIgE to rPen a 1 was detected in 98% of these patients. Of the 18 shrimp-tolerant patients, 61% had positive SPT to shrimp, 55% were IgE-positive to shrimp, and 33% showed IgE antibodies to rPen a 1. Determination of IgE to rPen a 1 yielded a positive predictive value of 0.72 and a negative predictive value of 0.91.

Conclusion:  IgE levels to rPen a 1 provided additional value to the diagnosis of shrimp allergy. Some allergens in mite extract are recognized by patients who are allergic to shrimp, though their clinical relevance remains unknown.

Abbreviations
SPT

skin prick test

DBPCFC

double-blind, placebo-controlled food challenges

SAP

clinically diagnosed shrimp-allergic patients

STP

shrimp-tolerant patients

DMP

dust-mite patients

SME

Solenocera melantho extract

SMST

Solenocera melantho shrimp tropomyosin

DPE

Dermatophagoides pteronyssinus extract

Shellfish allergy, including allergy to crustaceans and mollusks, is a relatively common and long-lasting disorder (1). Patients coming into contact with shellfish can experience a number of symptoms ranging from mild (e.g., allergy oral syndrome) to severe (e.g., anaphylaxis). Shrimp is the most prominent of all seafood because it is the most widely consumed and it causes the most severe reactions, even after cutaneous contact or inhalation.

The first major allergen identified in crustaceans was the muscle protein tropomyosin. Sensitization to shrimp tropomyosin, considered the primary allergen in shrimp, has been found in 80% of patients tested (2, 3). Tropomyosin is a 37-kDa protein associated with the actin filament of muscle cells as well as other noncontractile cells. Isoforms have been identified in several shrimp species, including Penaeus aztecus (Pen a 1), Penaeus indicus (Pen i 1), Penaeus monodon (Pen m 1), Metapenaeus ensis (Met e 1), and Litopenaeus vanamei (Lit v 1), with sequence identity ranging from 93% to 99%.

Tropomyosin is also considered to be responsible for cross-reactivity between other arthropods such as dust mites or cockroach (1, 4, 5). In tropomyosins from dust mites and other arthropods, sequence identity is about 75–80% (6). Therefore, the study of cross-reactivity between different tropomyosins can be useful in understanding this disorder.

Additional crustacean allergens have been characterized: the two minor cross-reactive crustacean allergens, arginine kinase (7, 8) and sarcoplasmic calcium-binding protein (9); myosin light chain, a new major shrimp allergen (10); troponin C; and triosephosphate isomerase. A complete list of shrimp allergens is available at allergen databases including http://www.allergen.org and http://www.allergome.com.

Despite several articles on shrimp allergens have been published in recent years, more data and tools are required to offer accurate diagnosis and understand the evolution and prognosis of shellfish allergy. In daily clinical practice, IgE to shrimp tropomyosin Pen a 1 may be measured by fluoroimmunoassay (ImmunoCAP system; Phadia, Uppsala, Sweden). In addition, IgE to a panel of shrimp tropomyosins Pen a 1, Pen i 1, and Pen m 1 can be assessed using the ImmunoCAP ISAC, a microarray-based semi-quantitative IgE testing system which has recently become commercially available. However, IgE to tropomyosin seems to have limited specificity for shrimp allergy diagnosis (4). One of the aims of this study is to evaluate the pattern of recognition of different tropomyosins in two subsets of well-characterized individuals: patients with shrimp allergy and patients with good tolerance to shrimp, as determined by double-blind, placebo-controlled food challenges (DBPCFC), by means of IgE analysis using natural and recombinant allergens.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Authors’ contributions
  8. Conflict of interest
  9. References

Design

The study was approved by the hospitals’ ethics committees, and all patients/guardians provided prior written informed consent.

Patient selection

A total of 45 patients were recruited from the Allergy Departments of two hospitals (FJD and Niño Jesús). Thirty-six had a history of immediate symptoms after ingestion of shrimp that were suggestive of shrimp allergy; some were sensitized to house dust mites. Nine patients with persistent rhinitis and/or asthma presenting positive skin prick test (SPT) and/or sIgE to mites were also included. All showed tolerance to shrimp.

Skin prick tests

Skin prick tests with commercial extracts of Dermatophagoides pteronyssinus (ALK-ABELLÓ, Madrid, Spain) and shrimp (Leti, Madrid, Spain) and prick-by-prick tests with raw red shrimp (Solenocera melantho) were performed according to European guidelines (10).

Food challenges

Double-blind, placebo-controlled food challenges were performed in all patients except those confirming well-tolerated intake of shrimp in the previous 3 months. Blinded challenges consisted of increasing doses of active and placebo puddings prepared by a hospital dietician. The pudding consisted of a milkshake made with vanilla and chocolate ice cream. S. melantho, one of the most widely consumed shrimp species in Spain, was added to the active meal. Five investigators confirmed adequate blinding.

None of the subjects were lactose intolerant or allergic to any compound of the challenge material. The challenge was performed at random. Challenges were considered positive if the patients experienced identical subjective symptoms (e.g., oral allergy syndrome) to three consecutive doses. In case of a negative DBPCFC, subjects were submitted to an open food challenge, receiving a total of 12 whole-cooked shrimp. The challenge procedure was halted if a definitive reaction was provoked.

Red shrimp (S. melantho), mite (D. pteronyssinus) extracts, natural, and recombinant purified tropomyosins

Solenocera melantho extract (SME) was prepared as described by Ayuso et al. (9).

Dermatophagoides pteronyssinus extract was provided by Leti SA, Spain. The protein concentration was estimated according to the method of Bradford (11).

Purified natural tropomyosin from P. monodon (nPen m 1) and purified recombinant tropomyosin from D. pteronyssinus (rDer p 10) were purchased from Bial, Bilbao, Spain.

Measurement of sIgE

Specific IgE antibodies to a commercial extract of shrimp, to D. pteronyssinus, and to rPen a 1 were measured by fluoroimmunoassay (ImmunoCAP; Phadia).

SDS-PAGE and immunoblotting

Crude SME, Dermatophagoides pteronyssinus extract (DPE), nPen m 1, and rDer p 10 were separated by SDS-PAGE as described previously (12). For immunodetection of IgE-binding proteins, the separated proteins were electroblotted onto a nitrocellulose membrane (Bio-Rad, Richmond, CA, USA) and incubated for 18 h at 4°C with a 1/3 dilution of the sera. Bound IgE antibodies were detected using peroxidase-conjugated rabbit anti-human IgE (DakoCytomation, Glostrup, Denmark) and using the ECL chemiluminescence method as recommended by the manufacturer (Amersham Biosciences, Buckinghamshire, UK).

Inhibition assays

For IgE immunoblotting inhibition, a sera pool from patients 2, 7, 14, and 16 was mixed with different concentrations of inhibitor solution. After incubation at 4°C for 16 h, the mixture was used as a primary antibody. The inhibitors employed were SME, nPen m 1, rDer p 10, and bovine serum albumin (negative inhibitor). Membranes containing SME, nPen m 1, and rDer p 10 were blocked and then exposed to preabsorbed serum. Bound IgE antibodies were detected as described above.

Data analysis

Subjects’ characteristics were described using descriptive statistics and expressed as mean and standard deviation. Normality was tested using the Kolmogorov–Smirnov test. Comparisons of mean values between groups were made using the Wilcoxon signed-rank test, the Kruskal–Wallis test, and the Mann–Whitney U-test and anova and unpaired t-test with Welch correction or t-test for nonparametric data and parametric data, respectively.

Associations were assessed by Pearson’s correlation; chi-squared test and anova were used on categorical and continuous variables, respectively. Differences were considered to be significant when P ≤ 0.05.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Authors’ contributions
  8. Conflict of interest
  9. References

Patients

Clinical results are summarized in Table 1. Thirty-six individuals with suspected reaction to shrimp and nine patients sensitized to house dust mites were included. Mean age was 23.9 ± 13.9 years. Twenty-two were men and 23 women.

Table 1.   Clinical characteristics of the patients, symptoms on challenge, skin tests to shrimp, and IgE measurements
Patient numberSexAge (years)Clinical presentationClinical presentationShrimp DBPCFCDefinitive diagnosisSPTSpecific IgE to shrimp kU/lSpecific IgE to Pen a 1 kU/lSpecific IgE to Dermatophagoides pteronyssinus kU/lIgE to shrimp tropomyosin by immunoblotIgE to nPen m 1 by immunoblotIgE to rDerp10 by immunoblotIgE to Dermatophagoides pteronyssinus by immunoblot
ShrimpDust mite
  1. Pathology: SF, shellfish; DM, dust mite.

  2. Symptoms: U, urticaria; R, rhinitis; RC, rhinoconjunctivitis; OAS, oral allergy syndrome; GI, gastric intestinal; A, asthma; C, cutaneous (rash, erythema); AE, angioedema; AN, anaphylaxis.

  3. Shrimp oral provocation: P, positive; N, negative; NI, not indicated; ND, not done.

  4. SPT, Skin prick test; P, positive; N, negative.

  5. Definitive Diagnosis: P, positive; N, negative.

  6. IgE to different tropomyosins by immunoblot: P, positive; N, negative; ND, not done.

  7. DBPCFC, Double-blind, placebo-controlled food challenges.

 1M33SF, DMOAS, GIRCPPP2.720.4613.90PPPP
 2F33SF, DMANRC, ANIPP100.0>10046.0PPPP
 3F28SF, DMR, A, URC, ANIPP7.032.03NDPPPN
 4M33SF, DMANRC, ANIPP6.152.832.27PPPP
 5F16SF, DMGIRCPPP5.244.3426.70PPPP
 6M48SF, DMR, GIR, APPP2.47<0.350.82PNNP
 7M8SF, DMUANIPP>100>10014.1PPPP
 8F43SF, DMURNIPP0.84<0.350.79PPPN
 9M13SF, DMOAS, GI, URCPPP5.50.792.15PPPP
10M5SF, DMURCPPP50.440.310.3PPPP
11M3SF, DMOAS, URCPPP13.77.850.86PPPP
12M14SF, DMAAPPP24.216.610.5PPPN
13F26SFAN NIPP5.602.60<0.35PPPP
14F7SFU NIPP>10061.811.1PPPND
15M9SFU, AE PPP11.79.485.75PPPN
16F15SFRC, U PPP96.795.518.7PPPND
17M7SFRC PPP0.60.41<0.35PPPN
18M4SFU NDPP39.825.84.57PPPN
19F36SF, DMU, RRC, ANNN<0.35<0.351.19PNNP
20F29SF, DMURCNNP0.44<0.350.75PPNN
21F12SF, DMOAS, URCNDNP5.98<0.351.56NNNP
22F18SF, DMOASRCNNP0.550.381.26NPPP
23F41SF. DMRCRCNNP0.48<0.359.75NNNP
24M36SF, DMRRC, ANNP<0.35<0.352.03NNNP
25M31SFU NNP0.62<0.350.46NNNN
26F32SFU, GI NNN<0.35<0.35<0.35NNNP
27M47SFC, AE NNN<0.35<0.35<0.35NNNN
28M26SFU, D, R NNN<0.35<0.35<0.35NNNN
29F24SFU NNN<0.35<0.35<0.35NNNN
30F40SFGI NNP<0.35<0.350.52NNNP
31F39SFU NNN<0.35<0.350.66NNPP
32M5SFRC NDNN0.810.480.68PPPN
33F24SFU NNP2.150.71<0.35PPPP
34M7SFGI NDNP15.48.621.94PPPN
35M7SFU, AE NNP16.510.12PPPN
36F2SFU NDNP0.410.38<0.35NNNND
37F43DM RCNNN<0.35<0.35<0.35NNNN
38M24DM RC, ANNN<0.35<0.353.75NPNN
39M26DM RCNNN5.77<0.35NDNNNP
40M15DM RCNNN2.76<0.358.56NNNN
41F52DM RCNNN<0.35<0.350.41NNNN
42M26DM RCNNN<0.35<0.355.68NNNP
43F27DM RCNNN<0.35<0.352.36PPNN
44F34DM RCNDNN5.92<0.358.16NNNN
45F29DM ANDNP1.45<0.3560.2NNNP

Shrimp allergy was assessed by DBPCFC for all food-allergic patients. Exceptions included seven cases determined by the clinicians from the two centers to be based on a medical history of severe systemic reaction upon shrimp ingestion, and another seven patients considered negative despite refusal of the challenge because they had recently tolerated shrimp in accidental ingestion. Patient 18 had developed immediate urticarial lesions after ingestion of shrimp on three occasions, and thus, he was considered positive.

According to the symptoms and the results of the DBPCFC, we classified the patients into three groups: shrimp-allergic patients (SAP), (n = 18); shrimp-tolerant patients (STP) (n = 18); and dust-mite patients (DMP) (n = 9). In the SAP group, 12 of 18 patients also had respiratory symptoms when exposed to dust mites, and six of 18 patients in the STP group experienced respiratory symptoms when exposed to dust mite.

Sensitization to shrimp, dust mites, and tropomyosins

The percentage of positive SPTs to shrimp across the different groups appears in Fig. 1A. All patients with clinical allergy to shrimp showed positive SPT (100%). In contrast, 61% (Specificity 0.5) of the STP and 11% of DMP had positive results, thus reaching statistical significance (P < 0.005 and P < 0.01, respectively).

image

Figure 1.  Percentage from different clinical analyses: Skin prick test (SPT) and specific IgE (sIgE) to shrimp extract, rPen a 1, and D. pteronyssimus as detected by fluoroimmunoassay. (A) Percentage of positive SPT and sIgE to shrimp extract, Pen a 1, and D. pteronyssimus. Significant differences are indicated in the figure. (B) Median levels of sIgE from the shrimp-allergic patients, shrimp-tolerant patients, and dust-mite patients groups. Significant differences are indicated in the figure.

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Specific IgE to shrimp extract was detected in all SAP cases (100%), as compared with 55% in STP (specificity = 0.54) and 44% in DMP. These differences are significant (P < 0.005, Fig. 1A). Also, the mean sIgE level in the SAP group shows significantly higher values than the STP (31.8 ± 9.3 vs 2.4 ± 1.2 kU/l, P < 0.005) group (Fig. 1B).

Specific IgE to rPen a 1 was detected in 16 patients (89%) in the SAP group (mean 26.1 ± 8.7 kU/l). Six STP (33%) showed positive sIgE to rPen a 1 (mean 1.15 ± 0.7 kU/l, P < 0.05) (Fig. 1A,B), with sensitivity of 0.88, specificity 0.77, positive predictive value 0.72, and negative predictive value 0.91.

Sixteen patients (89%) in the SAP group and 12 patients (67%) in the STP group had sIgE to D. pteronyssinus. Mean sIgE to DPE was 9.4 ± 2.8 vs 1.3 ± 0.5 kU/l (P < 0.005, Fig. 1A,B).

Four patients (44%) in the DMP group had sIgE to shrimp, seven (87%) to D. pteronyssinus (in one subject this test was not performed, though all had positive SPT to D. pteronyssinus), and none to rPen a 1 (Fig. 1A). There was a significant correlation between values of IgE to shrimp and to rPen a 1 (r = 0. 978 P < 0.01) as well as values of IgE to rPen a 1 and to D. pteronyssinus (r = 0. 711 P ≤ 0.01) and to shrimp and to D. pteronyssinus (r = 0. 675 P ≤ 0.01).

SDS-PAGE and immunoblotting

SDS-PAGE of SME and DPE showed several protein bands within a molecular weight (MW) ranging between 7 and 97 kDa. The most prominent bands in the SME were around 21–24, 35, and 37–39 kDa. With respect to purified nPen m 1 and rDer p 10, a prominent band at 37 kDa was observed (Fig. 2B).

image

Figure 2.  (A) Solenocera melantho picture; (B) SDS-PAGE. SDS-PAGE of S. melantho extract (lane 2), Dermatophagoides pteronyssinus extract (lane 3), purified natural tropomyosin from Penaeus monodom (Pen m 1) (lane 4), and purified recombinant tropomyosin from D. pteronyssinus (Der p 10) (lane 5). Lane 1, molecular weight markers. The contents of loaded protein were 10 μg. The protein in SDS-PAGE was stained with colloidal Coomassie blue.

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The IgE-binding ability of SME (Fig. 3A), D. pteronyssinus (Fig. 3B), nPen m 1 (Fig. 3C), and rDer p 10 (Fig. 3D) was evaluated by immunoblotting. All SAP (100%) showed IgE binding to a protein around 37–39 kDa, which is consistent with tropomyosin, the major allergen of shrimp. This allergen was recognized by 33.3% in the STP group and by only 11% of DMP (Fig. 4). All SAP exhibited IgE reactivity to several shrimp proteins (Fig. 3A).

image

Figure 3.  IgE reactivity. IgE reactivity of Solenocera melantho extract (3A), Dermatophagoides pteronyssinus extract (3B), nPen m 1 (3C), and rDer p 10 (3D) under reducing conditions. IgE reactivity to tropomyosin is represented at 37–40 kDa. Control immunoblot experiments with the sera from the nonatopic patients did not show any IgE-binding bands.

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image

Figure 4.  Percentage of positive IgE reactivity to 37- to 39-kDa Solenocera melantho extract bands, Pen m 1, Der p 10, and Dermatophagoides pteronyssinus from three groups of patients. For 37- to 39-kDa SME bands and Pen m 1, the percentage of recognition in shrimp-allergic patients (SAP) is higher than in the shrimp-tolerant patients (STP) and dust-mite patients groups. The percentage of recognition to Der p 10 shows significant differences between the SAP and STP groups. Among the three groups, there are no significant differences in recognition to 37- to 39-kDa D. pteronyssinus bands.

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By contrast, the IgE reactivity to DPE was similar across all groups. In DPE, the protein around 37–39 kDa, which corresponds to the tropomyosin of D. pteronyssinus, was recognized by 10/16 SAP (62.5%), 9/17 (53%) STP, and 3/9 (33%) DMP. These data showed nonsignificant differences between the three groups (Fig. 4). Among the proteins of DPE recognized by the IgE of patient sera, a 20-kDa protein band, corresponding to the major allergen Der p 2 of dust mite, was recognized by the three groups.

Among SAP, 94% showed IgE reactivity to nPen m 1 and rDer p 10 (Fig. 4); 33% of patients in the STP group recognized nPen m 1 and rDer p 10 t (Fig. 4). In the DMP group, only 22% showed IgE reactivity to nPen m 1, and no IgE binding was seen with rDer p 10 (Fig. 4).

Cross-reactivity study

Inhibition of the S. melantho IgE-binding proteins was already relevant when the sera pool was preincubated with 10 μg of nPen m 1, and inhibition was nearly total with 100 μg of nPen m 1 as inhibitor (Fig. 5A). Nevertheless, when the inhibitor extract was S. melantho, only 50 μg was necessary to reach total inhibition (Fig. 5B).

image

Figure 5.  Immunoblot inhibition. Immunoblot inhibition of IgE to Solenocera melantho by Pen m 1 and Der p 10 (A). Immunoblot inhibition of Pen m 1 IgE by S. melantho extract (SME) and Der p 10 (B). Immunoblot inhibition of Der p 10 IgE by SME and Pen m 1 (C). Sera were preabsorbed with different quantities (μg) of SME or tropomyosins as described on top of each figure. Lane 0 corresponds to preincubation of sera with 50 μg of bovine serum albumin. C represents control sera of a nonatopic subject.

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Cross-inhibition studies between SME and rDer p 10 revealed that 100 μg of rDer p 10 was not enough to achieve full inhibition of IgE binding to tropomyosin of SME (Fig. 5A), and the preincubation of the sera pool with an equal concentration of SME led to an absence of IgE reactivity to the rDer p 10-immobilized protein (Fig. 5C).

Preabsorption of the sera pool with 50 μg of nPen m 1 induced complete inhibition of IgE binding to immobilized rDer p 10 (Fig. 5C), and when rDer p 10 was used as the inhibitor allergen, total inhibition of IgE binding to immobilized nPen m 1 was not possible at a dose of 50 μg (Fig. 5B), or with 100 μg of this allergen (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Authors’ contributions
  8. Conflict of interest
  9. References

Crustaceans – particularly shrimp – are highly allergenic foods which are responsible for food-induced reactions in both children and adults (13, 14). Diagnostic testing tools for food hypersensitivity have improved over time, but both in vivo and in vitro methods still fall short of producing accurate diagnosis (15). Double-blind, placebo-controlled food challenges is still the gold standard for the diagnosis of food allergy because it avoids false-positive diagnoses (16, 17). In our study, only 18 of 36 patients with symptoms suggestive of shrimp allergy had confirmed diagnosis of clinical allergy to shrimp; indeed, it was necessary to perform 24 DBPCFCs because prick-prick or sIgE to shrimp was positive in 28 patients. Oral challenges always entail a risk and are time-consuming. Therefore, any tool that helps clinicians avoid this procedure is welcome. Some studies have suggested high-risk cutoffs of sIgE to cow’s milk, egg, peanut, soy, and wheat (18, 19), but no data have been published on shrimp to date.

In our population, SPT to shrimp was positive in 100% of the SAP group and in 61% of the STP group, a finding that corroborates the high sensitivity of this test but its low specificity (around 50%). Similar levels of sensitivity are maintained when measuring sIgE to rPen a 1, but its specificity was higher (77%vs 50%). Our study failed to determine a sIgE cutoff value for shrimp extract which was able to predict shrimp challenge results. However, patients from the SAP group had significantly higher values of sIgE to shrimp. Therefore, diagnosis of shrimp allergy is still a challenge for clinicians, particularly for patients with positive skin test or sIgE to shrimp with or without previous reaction to shrimp.

It has recently been reported that detection of IgE to shrimp tropomyosin is linked to the presence of clinical shrimp allergy in a Brazilian population (20). The authors found that a positive history associated with a positive result for IgE to shrimp tropomyosin resulted in positive oral challenge in 100% of patients, but two of eight tropomyosin-negative patients had positive oral challenge to shrimp. Likewise, a negative history associated with a negative test result for IgE to shrimp tropomyosin was always associated with a negative challenge result. We obtained similar results when measuring IgE to rPen a 1 by fluoroimmunoassay, as results were positive in all the SAP except 2 (6 and 8). Patient 6, despite being negative to rPen a 1 obtained by fluoroimmunoassay and to nPen m 1 by immunoblotting, presents sIgE to shrimp extract under both techniques. We therefore cannot rule out the possibility that the patient’s shrimp allergy was because of sensitization to other allergens like arginine kinase, whose MW resembles that of tropomyosin. As seen here, it would be beneficial to have the whole panel of identified shrimp allergens available for testing. Moreover, identification of allergenic epitopes by microarray would help define the risk level of being clinically allergic (21).

This study characterizes S. melantho shrimp as an allergenic source, and the data suggest that S. melantho presents tropomyosin as an important allergen. Further studies including purification strategies and sequencing would be necessary to better characterize Solenocera melantho shrimp tropomyosin (SMST) and other relevant allergens from this source.

Owing to high cross-reactivity between nPen m 1 and SMST, we define a potential use of different recombinant tropomyosins as a marker of sensitization and clinical reactivity.

Cross-reactivity among tropomyosins from dust mite and S. melantho shrimp is demonstrated in this study based on inhibition assays using pooled sera from SAP. It has been suggested that tropomyosin is a cross-reacting allergen between foods and aeroallergens of animal origin, such as dust mite or cockroach. IgE reactivity to shrimp has been demonstrated in an unexposed population of subjects allergic to mite and/or cockroaches (5), and, conversely, a study has suggested that sensitization to shrimp tropomyosin can cause allergy to mite and Blattella germanica (22). Our results from inhibition assays indicate that similar epitopes of S. melantho shrimp and dust-mite tropomyosin are involved in IgE recognition, but further studies on the peptide sequence and tertiary structure of common epitopes should further elucidate the molecular mechanism of IgE cross-reactivity (4, 23).

In the SAP group, there are more subjects with respiratory allergy to dust mite (12/18) than in the STP group (6/18); this could be attributed to the role of several proteins that were recognized by the patients’ IgE as observed by immunoblotting with SME and DPE. None of the patients with respiratory allergy to dust mites have positive results detected by fluoroimmunoassay to rPen a 1; however, in two of nine subjects in this group, IgE-binding bands were detected by immunoblotting to nPen m 1, but no patients presented IgE binding to rDer p 10. The failure to recognize Der p 10 is surprising when considering positive nPen m 1; however, the sequence identity of shrimp tropomyosin and Der p 10 is approximately 80%. It may be possible that these molecules have species- sIgE epitopes. This idea is supported by the fact that Der p 10 inhibited only partially the IgE binding of the sera pool to shrimp tropomyosins. It could be beneficial to do the same analysis on a patient-by-patient basis.

In most DMP living in Europe and in the United States, sensitization to mite tropomyosins is not important to the sensitization process (24–28). Here, we must highlight that tropomyosin appears in low concentration in mites (29). Even though lower levels of this protein are present in mites than in crustaceans, a study in Africa revealed that IgE recognition frequency to rDer p 10 can be significant (55%), which may reflect sensitization to the cross-reactive allergen tropomyosin present in parasites (30).

In conclusion, this study demonstrates that measurements of IgE levels to shrimp tropomyosin (rPen a 1) provide added diagnostic value to the methods currently available – SPT or sIgE to shrimp – for confirmation of shrimp allergy in patients with suspected reactions in a European population. Nevertheless, we must keep in mind that specificity of IgE to rPen a 1 is 0.77; thus, oral challenge testing may be necessary in some patients. Furthermore, we reported SMST as an allergen, demonstrating its high cross-reactivity with mite tropomyosin.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Authors’ contributions
  8. Conflict of interest
  9. References

This study was supported by Mútua Madrileña, Red RESPIRA C03/011, CIBER grants from Spain’s Health Research Fund (Fondo de Investigaciones Sanitarias– FIS), and SEAIC (Sociedad Española de Alergia e Inmunología Clínica).

Authors’ contributions

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Authors’ contributions
  8. Conflict of interest
  9. References

VdP and JS conceived of the study, designed and planned the experiments, participated in the analysis of the data, and wrote significant sections of the manuscript; CG carried out the immunological study, performed the analysis and interpretation of the data, and helped to write the manuscript; patients’ recruitment and diagnosis were performed by SG, and MDI helped to draft the manuscript; EA and RL performed SDS-PAGE; EL and BS performed the inhibition procedures. All authors read and approved the final manuscript.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Authors’ contributions
  8. Conflict of interest
  9. References

Joaquín Sastre is consultant to Phadia, Mundipharma, Merck, GSK, and Stallergens; having been paid lecture fees by Novartis, GSK, Stallergenes, Merck, and UCB; and having received grant support from Phadia, GSK, and ALK-Abello. The rest of authors declare that they have no competing interests.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Authors’ contributions
  8. Conflict of interest
  9. References