Tropomyosin is a minor but distinct allergen in patients with shrimp allergies in Japan

Recently, purified allergens have been utilized for allergen‐specific IgE tests, which are highly useful because of their excellent specificity and sensitivity in identifying allergic patients. The purpose of this study was to evaluate the specificity and sensitivity of tropomyosin‐specific IgE test in the diagnosis of shrimp allergies in Japan. We enrolled 27 patients with shrimp allergy and five patients with atopic dermatitis, who had no history of allergic reactions to shrimp but showed positive results in tropomyosin‐specific IgE test, in this study. Tropomyosin‐specific IgE was determined by IgE immunoblotting and tropomyosin‐specific IgE test. Involvement of carbohydrate moieties in IgE binding to the allergens was examined by periodate treatment. Tropomyosin‐specific IgE was detected in 13 and positive in 10 of the 27 patients with shrimp allergy, whereas shrimp‐specific IgE was detected in 21 and positive in 20 of these 27 patients. Of the 13 patients with detectable levels of tropomyosin‐specific IgE, seven were confirmed to have tropomyosin‐specific IgE by immunoblotting analysis, whereas no IgE binding was seen in the five patients with atopic dermatitis, indicating the high specificity of the tropomyosin‐specific IgE test. The level of tropomyosin‐specific IgE was well correlated with those of shrimp‐specific IgE and Der p 10‐specific IgE. Our findings indicated tropomyosin is a minor but distinct allergen in patients with shrimp allergy, especially causing symptoms of OAS.

allergy have IgE to tropomyosin. 2,3 Tropomyosin was identified as a major allergen in other crustaceans such as lobster Homarus americanus (Hom a 1) 4 and crab Charybdis feriatus (Cha f 1), 5 mollusks such as squid Todarodes pacificus (Tod p 1), 6 oyster Crassostrea gigas (Cra g1), 7 and snail Turbo cornutus (Tur c 1). 8 In addition, many reports present tropomyosin to be an important component of immune and allergic reactions in other invertebrates such as house dust mites (HDMs) Dermatophagoides farinae (Der f 10), 9 Dermatophagoides pteronyssinus (Der p 10), 10 and cockroach Periplaneta Americana (Per a 7). 11 Tropomyosins from HDMs have a high sequence homology to shellfish tropomyosins, with an 81% amino acid sequence similarity between shrimp (Pen a 1) and HDMs (Der p 10), 12 and cross-reactivity between tropomyosins from shellfish and HDMs has also been well documented. 13,14 These findings provided evidence for cross-reactivity in allergen-specific IgE test among crustaceans, mollusks, insects, arachnids, and even nematodes. The elucidation of the amino acid sequences of tropomyosin protein in these species is important to understand IgE cross-reactivity among tropomyosins of different species.
Allergen-specific IgE test is widely used in the diagnosis of immediate-type food allergies because this test can be performed with ease for identifying causative allergens in patients with food allergies. It is noteworthy that the in vitro immunoassay system usually employs crude extracts of foodstuffs to detect the food-specific IgE; thus, the sensitivity and specificity of the test are not always satisfactory in identifying patients with true food allergies. Recently, purified allergens have been utilized for allergen-specific IgE tests; these tests show excellent specificity and sensitivity in identifying allergic patients and are termed component-resolved diagnostics. [15][16][17][18][19] The purpose of this study was to evaluate the specificity and sensitivity of tropomyosinspecific IgE test in the diagnosis of shrimp allergies in Japan.

| Measurement of serum allergen-specific IgE
Sera obtained from the patients were frozen and stored at À20°C until use. Serum shrimp-specific IgE, crab-specific IgE, squid-specific IgE, tropomyosin-specific IgE, Der p 1 (peptidase 1)-specific IgE, and Der p 10 (tropomyosin)-specific IgE levels were detected by the CAP-fluorescent-enzyme immunoassays (CAP-FEIA) (ImmunoCAP â , ThermoFischer Scientific, Uppsala, Sweden). Values greater than 0.35 Ua/mL were considered to be detectable and those greater than 0.70 Ua/mL were considered to be positive. IgE immunoblotting was performed as previously described. 20 Briefly, proteins were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples (shrimp extract: 6 lg/lane, purified tropomyosin: 0.5 lg/lane) were loaded on a 12.5% polyacrylamide gel and electrophoresed at 250 V for one hour. The separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P â ; Millipore, Billerica, MA, USA) using a semi-dry immunoblot apparatus (Bio-rad) at 25 V for one hour. After the transfer was completed, the membrane was

| Sodium periodate treatment
Binding of IgE to carbohydrate moieties of shrimp protein was examined by treating the blotted PVDF membranes with sodium periodate treatment according to a method previously described. 20 Briefly, water-soluble and water-insoluble shrimp proteins were separated on 12.5% SDS-PAGE under reducing condition and electrophoretically transferred to a PVDF membrane. After the blotting, PVDF membranes were incubated with 0.02 mol/L NaIO4 in 0.05 mol/L sodium acetate (pH 5.0) for one hour at 28°C in the darkness. The membranes were then immunoblotted as described above. The blotted PVDF membrane immersed in 0.05 mol/L sodium acetate (pH 5.0) served as the control.

| Statistical analysis
Levels of allergen-specific IgE were compared using Pearson correlation test and P-value with < 0.05 was considered to be significant.

Statistical analysis was performed with SPSS Statistical version 24
(IBM Corporation, Tokyo, Japan). Clinical symptom: OAS, oral allergy syndrome; U, urticaria; CU, contact urticaria; C, cough; As, asthma; AE, angioedema; AP, abdominal pain; F, fever; R, respiratory; RD, respiratory discomfort; S, shock. c AD: atopic dermatitis patient who has no history of allergic reaction to shrimp but has positive shrimp-specific IgE test.

| Allergen-specific IgE test
Shrimp-specific IgE was detected in 21 (detection rate 77.8%) and positive in 20 (positivity rate 74%) of the 27 patients with shrimp allergy, whereas tropomyosin-specific IgE was detected in 13 (detection rate 48.1%) and positive in 10 (positivity rate 37%) of these 27 patients (Table 2). When the patients were divided into three groups according to their symptoms, the shrimp-specific IgE was detected in 10 (detection rate 91%) and positive in 10 (positivity rate 91%) of the 11 patients in group I, detected in 8 (detection rate 61.5%) and positive in eight (positivity rate 61.5%) of the 13 patients in group II, and detected in three (detection rate 100%) and positive in 2 (positivity rate 67%) of the three patients in group III ( In the five patients with AD, without any history of allergic reaction to shrimp, tropomyosin-specific IgE test was detected in three subjects (detection rate 60.0%) and positive in one subject (positivity rate 20.0%).
In the positive tests for the 27 patients with shrimp allergy and five patients without shrimp allergy, sensitivity of shrimp-specific IgE test was 74% but specificity was 0%, whereas sensitivity of tropomyosin-specific IgE test was 37% but specificity was 80%.

| IgE immunoblotting
Serum Serum IgE binding to purified tropomyosin was detected in the seven patients with shrimp allergy (subjects 2, 4, 6, 11, 21, 23, and 26), whereas no binding was seen in the five patients with AD (Figure 2). The binding was well compatible with the binding to 34-35 kDa bands seen with shrimp extract proteins, indicating that these bands predominantly arose from tropomyosin.

| Sodium periodate treatment
We performed periodate oxidation of sugar residues to determine whether carbohydrate epitopes were involved in the IgE reactivity of shrimp extract. In the subjects 2, 6, 11, 21, and 23, individual bands with molecular weight of 34-35 kDa were not affected. However, the 80-kDa band found in subject 6 and 43-kDa band found in subject 29 was diminished, suggesting IgE binding to carbohydrate moieties in these bands (Figure 3).

| Correlation among allergen-specific IgE levels
The level of tropomyosin-specific IgE strongly correlated to that of Der p 10-specific IgE, but not to that of Der p 1-specific IgE (Figure 4). The level of tropomyosin-specific IgE strongly correlated to that of shrimp-specific IgE, whereas the level of Der p 10-specific IgE did not correlate to that of Der p 1-specific IgE ( Figure 5).

| DISCUSSION
In this study, we demonstrated that tropomyosin is a minor but distinct allergen in the patients with shrimp allergy in Japan, because, in F I G U R E 1 IgE immunoblot analysis using water-soluble and water-insoluble shrimp proteins. A, Gel stained with Coomassie Brilliant Blue. B, Immunoblotting with the subjects' sera. Lane M: molecule weight marker proteins; Lane S: water-soluble shrimp proteins; I: water-insoluble shrimp proteins spite of its low sensitivity (37%), the specificity of the tropomyosinspecific IgE test (80%) was much higher than that of the shrimp-specific IgE test (0%). In addition, the findings via immunoblotting were well compatible with the results of tropomyosin-specific IgE tests, supporting that tropomyosin is a highly specific allergen for shrimp allergy. The positive value of the tropomyosin-specific IgE test (1.02 Ua/mL) found in the patient with AD (subject 28) was a non-specific reaction, as no clear band was detected by immunoblotting. Furthermore, the 43-kDa band seen in another patient with AD (subject 29) was considered realistic because of the binding to the carbohydrate moieties irrelevant to tropomyosin, as the binding was diminished after periodate treatment, removing these moieties (Figure 3).
The positivity rate of tropomyosin-specific IgE test in the present study (37%) was far lower than those obtained in the previous studies: Ayuso et al, 21 Gamez et al, 22  only 41% and 15.8%, respectively, were found to be tropomyosinreactive when investigated by immunoblot analysis and tropomyosin-specific IgE measurements. 23,24 These findings indicate that sensitization to tropomyosin may be dependent on the geographic area, and low sensitization rates are observed in the areas located near the sea, such as Japan, Italy, and Singapore.  The reason why the two patients (subjects 23 and 26) showed 34-35 kDa band only in the water-insoluble proteins of shrimp was not clarified in this study, but we speculated that the amount of tropomyosin in the water-soluble fraction is less compared with that in the water-insoluble fraction, because tropomyosin is fundamentally water-insoluble protein. Cross-reactivity between crustaceans and mollusks is also clearly explained by the sensitization to tropomyosin because the nine patients with positive tropomyosin-specific test showed positive IgE against crab and squid. When we analyzed relation between level of tropomyosin-specific IgE and that of Der p 1specific IgE, a significant correlation was found, although no significant correlation was seen between level of tropomyosin-specific IgE and that of Der p 1-specific IgE ( Figure 4). This indicates that shrimp tropomyosin-specific IgE strongly cross-reacts to Der p 10. Here raises a question which is the first, sensitization to shrimp tropomyosin or sensitization to Der p 10. Then, we analyzed relation between level of Der p 1-specific IgE and that of Der p 10-specific IgE, in addition to relation between level of tropomyosin-specific IgE and that of shrimp-specific IgE ( Figure 5). As shown in the Figure 5, strong correlation was found between shrimp-specific IgE and tropomyosin-specific IgE, however, no significant correlation was seen between Der p 1-specific IgE and Der p 10-specific IgE, indicating that specific IgE sensitized to shrimp tropomyosin cross-react to HDM tropomyosin.
In the present study, we detected IgE bands in seven of the 27 (26%) patients with shrimp allergy using immunoblotting, whereas the shrimp-specific IgE test was detected in 21 of these 27 patients.
In addition, we detected tropomyosin-specific IgE only in 13 of the 27 patients with shrimp allergy, suggesting that there are still undetermined shrimp allergens in other patients and more sensitive tests are required to identify patients with shrimp allergy in Japan. The reason why we found low prevalence of tropomyosin-sensitization in our patients compared with those of the overseas-reports is not clearly elucidated in this study. We speculate that most of the Japanese people would be tolerant to shrimp tropomyosin because they often eat shrimp since childhood, compared with the USA, European, and Brazilian populations who have high sensitization rate to shrimp tropomyosin.