Snail allergy is considered a serious form of food allergy with prevalence of 1.4 to 22% worldwide. However, allergy to Pomacea canaliculata, a commonly consumed local snail has not been well-described. Hence, this study aimed to characterize the allergenicity and stability of the allergenic proteins of P. canaliculata by proteomics approach. Snail flesh was treated with several thermal and non-thermal treatments prior to overnight protein extraction. The snail proteins were then subjected to SDS-PAGE, followed by immunoblotting, two-dimensional electrophoresis (2-DE), 2-DE immunoblotting and mass-spectrometry analysis. Raw snail demonstrated 31 protein bands between 10 to 250 kDa, with fewer protein bands in treated snails. Boiled snails had the most protein bands among thermally treated snails, while salted and dried snails showed more bands than pickled snails among non-thermal treatments. Immunoblotting of raw extract demonstrated 16 IgE-binding bands, with the 33 and 42 kDa protein bands were identified as the major. The 33 kDa allergen was highly stable to all treatments applied, while the 42 kDa was sensitive to thermal and pickling treatments. Fewer allergenic bands were present in treated snails, with allergenicity ranked as raw > boiled > roasted > fried for thermal treatments, and raw > salted > dried > pickled for non-thermal treatments. Mass spectrometry identified the 33 kDa and 42 kDa allergens as tropomyosin and actin, respectively. In conclusion, P. canaliculata has numerous allergenic proteins with varying stability. This result is essential in facilitating the enhancement of global strategy for diagnosis and management of snail allergic patients worldwide.

BACKGROUNDSnail allergy is rare but can be fatal. Pila polita, a freshwater snail, was considered as a popular exotic food, particularly in tropical countries, and consumed in processed forms. Thus, the purpose of this study was to identify the major and cross-reactive allergens of P. polita and to determine the impact of food processing on the allergen stability. RESULTSSodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated raw snail extract to approximately 24 protein bands, between 9 and 245 kDa. The prominent band at 33 kDa was detected in all raw and processed snail extracts. Immunoblotting tests of the raw extract demonstrated 19 immunoglobulin E (IgE)-binding proteins, and four of them, at 30, 35, 42 and 49 kDa, were revealed as the major IgE-binding proteins of P. polita. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified the 49 and 42 kDa major allergens as actin, whereas the 30 and 35 kDa major allergens were identified as tropomyosin. Immunoblotting revealed that the raw snail had more allergenic proteins than the processed snail. The degree of allergenicity in decreasing order was raw > brine pickled> boiled > roasted > fried > vinegar pickled. The presence of cross-reactivity between P. polita and the shellfish tested was exhibited with either no, complete, or partial inhibitions. CONCLUSIONActin and tropomyosin were identified as the major and cross-reactive allergens of P. polita among local patients with snail allergy. Those major allergens are highly stable to high temperatures, acidic pH, and high salt, which might played a crucial role in snail allergy in Malaysia. (c) 2023 Society of Chemical Industry.

Background Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity. Methods and Results The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata . Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae , ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients. Conclusions This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

Crab allergy is reported as a serious form of food allergy in many countries. This study was aimed to identify the major allergens of the local mud crab, Scylla tranquebarica (S. tranquebarica), and subsequently, determine the effect of vinegar treatments on the crab allergens. Crab muscles were treated with synthetic and natural vinegar. Crab proteins were then extracted from the untreated and vinegar-treated crabs. All extracts were then fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by immunoblotting; using sera from crab-allergic patients. The crab proteins were then further fractionated by two-dimensional electrophoresis (2-DE) and analyzed by mass spectrometry (MS). The untreated crab had 38 protein bands, while that was only a few bands between 18 to 73 kDa for the vinegar-treated crabs. Immunoblotting of untreated crab revealed 20 IgE-binding bands, whereas the vinegar-treated crabs could only retain a few IgE-binding bands. Five major allergens were identified with molecular weights of 38, 42, 49, 63, and 73 kDa in the untreated crab. In contrast, the vinegar-treated crabs had only a few major allergens with molecular weights of 38, 42, and 73 kDa. MS identified the 43 and 49 kDa as arginine kinase, while the 38, 63, and 73 kDa were identified as tropomyosin, actin, and hemocyanin, respectively. In conclusion, we found three common major allergens for S. tranquebarica including tropomyosin, arginine kinase, and actin, and one novel allergen known as hemocyanin. All the major allergens could retain minimal allergenic capability in vinegar-treated crabs, suggesting that vinegar treatments might be useful to reduce crab allergenicity. These data would assist the clinicians in the management of crab-allergic patients worldwide.

Schistosomiasis is a tropical disease caused by fluke worms of genus Schistosoma. It can present as acute schistosomiasis and present as fever, cough, headaches, urticaria and cercarial dermatitis and this vary according to the infecting Schistosoma species (Jaureguiberry et al. 2010). Twenty-seven (27) year old female presented to the allergy clinic in Kuala Lumpur Hospital with chronic urticaria for past one year. She presented with persistent severe itchy maculopapular skin eruptions, mostly at the trunk and lower limbs, sparing the face and neck and not associated with angioedema. She had sought treatment from several general practitioners and dermatologists. She was treated with antihistamines, montelukast, and corticosteroid cream but she had temporary relief of her symptoms. She worked as an executive officer in a firm, and she has history of travelling to Cambodia and Thailand regularly for work. She gave history of eating raw salad and seafood in both places. Total Immunoglobulin E (IgE) was elevated (232ku/l). Low specific IgE to seafood. Parasitologic enzyme-linked immunosorbent assay (ELISA) test showed positive titer Immunoglobulin G (IgG) 0.311 to schistosomiasis. Urine microscopy and direct stool microscopy showed negative schistosomiosis eggs. Full blood picture was normal. Ultrasound of abdomen showed normal liver, spleen, kidney, and urinary system. She was given one dose of oral Praziquantel and antihistamines. For about a month, her urticaria and pruritus were less. After 6 months of treatment with praziquantel, a repeat parasitologic ELISA test to IgG was negative to schistosomiasis. Repeated urine microscopy and direct stool microscopy were negative to schistosomiosis. In summary, this woman had presented with chronic urticaria, with history of travelling to endemic areas for schistosomiasis and eating raw food perhaps with contaminated water, it is worthwhile to do some parasitic screening test for common parasitic helminthes, even though it is rarely associated with urticaria. However, cercarial dermatitis due to schistosomiasis have been reported by Folster-Holst R, et al (2001), S.J. Fraser et al (2008), and Kolarova L, et al (1999). To date, schistosomiasis is still considered as one of the main neglected tropical diseases by World Health Organization (WHO).

Prawn allergy is a common cause of allergic reactions in countries where crustacean is a famous seafood. Macrobrachium rosenbergii (giant river prawn) is declared as a local major food allergens, causes severe symptoms like asthma and anaphylactic shock. Therefore, the goal concerning this research is to identify the allergenicity of heat treated of M. rosenbergii. Prawn extracts have been prepared using fresh raw prawn and three heat-treated prawn flesh (boiled, steamed, fried), then afterward analyzed by the usage of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Allergenic proteins were identified by means of immunoblotting by the use of sera from 30 prawn-allergic patients. The identities of selective major allergens were then determined by mass-spectrometry analysis. The raw prawn has 27 protein fractions between 6 to 207 kDa, while the heat-treated prawns have reduced number of protein bands. Six prominent bands at 72, 65, 48, 38, 36, and 30 kDa were considered as the major allergens of raw extract. Meanwhile, after heat remedies applied, most of the IgE-binding bands were disappeared except for three major allergens at 38, 36 or 30 kDa which were still remain to be seen, suggesting as highly thermostable allergens. Among heat-treated prawns, steamed and boiled elicited more allergenic bands, contrast to fried prawn extract. Mass spectrometry analysis identified two selected major allergens at 36 and 48 kDa as tropomyosin and arginine kinase, respectively. As a conclusion, this study indicated that both thermostable and thermolabile proteins from M. rosenbergii were allergenic. Tropomyosin and arginine kinase were identified as the most important major allergens. Thus, the knowledge on thermostability of prawn allergens are crucial for improving diagnosis and management of prawn allergic patients in this county.

To investigate immunoglobulin E (IgE)-mediated hypersensitivity to chlorhexidine (CHX), and association between CHX exposure and serum specific IgE (SIgE) levels, among first-year dental students. Participants completed a questionnaire on medical health, history of allergies/hypersensitivities and CHX exposure. A sample of venous blood (3 mL) was drawn and subjected to SIgE test to CHX; sensitisation defined as SIgE level of ≥0.10 kUA/L. Fifty-eight (98.3%) participants, consisting of 69% (n = 40) female with a mean age of 21, were recruited; three quarters (84%) reported no known health issue, and over half had no history of allergies (57%); less than 20% reported having had exposure to CHX-containing products with the majority from toothpaste (9%) and mouthwash (12%). The CHX SIgE test showed that 8.6% (n = 5) were sensitised. No association between CHX exposure history and the level of SIgE antibody was noted. Although most participants reported no known exposure to CHX, 8.6% showed CHX sensitisation suggesting unknown exposure and the potential risk of developing hypersensitivity and adverse reactions in the future.

Prawn allergy is a common cause of allergic reactions in countries where crustacean is a famous seafood. Macrobrachium rosenbergii (giant river prawn) is declared as a local major food allergens, causes severe symptoms like asthma and anaphylactic shock. Therefore, the goal concerning this research is to identify the allergenicity of heat treated of M. rosenbergii. Prawn extracts have been prepared using fresh raw prawn and three heat-treated prawn flesh (boiled, steamed, fried), then afterward analyzed by the usage of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Allergenic proteins were identified by means of immunoblotting by the use of sera from 30 prawnallergic patients. The identities of selective major allergens were then determined by massspectrometry analysis. The raw prawn has 27 protein fractions between 6 to 207 kDa, while the heat-treated prawns have reduced number of protein bands. Six prominent bands at 72, 65, 48, 38, 36, and 30 kDa were considered as the major allergens of raw extract. Meanwhile, after heat remedies applied, most of the IgE-binding bands were disappeared except for three major allergens at 38, 36 or 30 kDa which were still remain to be seen, suggesting as highly thermostable allergens. Among heat-treated prawns, steamed and boiled elicited more allergenic bands, contrast to fried prawn extract. Mass spectrometry analysis identified two selected major allergens at 36 and 48 kDa as tropomyosin and arginine kinase, respectively. As a conclusion, this study indicated that both thermostable and thermolabile proteins from M. rosenbergii were allergenic. Tropomyosin and arginine kinase were identified as the most important major allergens. Thus, the knowledge on thermostability of prawn allergens are crucial for improving diagnosis and management of prawn allergic patients in this county.

Prawn allergy is a common cause of allergic reactions in countries where crustacean is a famous seafood. Macrobrachium rosenbergii (giant river prawn) is declared as a local major food allergens, causes severe symptoms like asthma and anaphylactic shock. Therefore, the goal concerning this research is to identify the allergenicity of heat treated of M. rosenbergii. Prawn extracts have been prepared using fresh raw prawn and three heat-treated prawn flesh (boiled, steamed, fried), then afterward analyzed by the usage of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conformity with their protein profiles. Allergenic proteins were identified by means of immunoblotting by the use of sera from 30 prawn-allergic patients. The identities of selective major allergens were then determined by mass-spectrometry analysis. The raw prawn has 27 protein fractions between 6 to 207 kDa, while the heat-treated prawns have reduced number of protein bands. Six prominent bands at 72, 65, 48, 38, 36, and 30 kDa were considered as the major allergens of raw extract. Meanwhile, after heat remedies applied, most of the IgE-binding bands were disappeared except for three major allergens at 38, 36 or 30 kDa which were still remain to be seen, suggesting as highly thermostable allergens. Among heat-treated prawns, steamed and boiled elicited more allergenic bands, contrast to fried prawn extract. Mass spectrometry analysis identified two selected major allergens at 36 and 48 kDa as tropomyosin and arginine kinase, respectively. As a conclusion, this study indicated that both thermostable and thermolabile proteins from M. rosenbergii were allergenic. Tropomyosin and arginine kinase were identified as the most important major allergens. Thus, the knowledge on thermostability of prawn allergens are crucial for improving diagnosis and management of prawn allergic patients in this county.

Background: Apart from the nutritional benefits, bivalves are also considered to be among a group of allergenic foods. Symptoms of allergy to bivalves are largely similar to those of allergic reactions reported for other shellfish. The identification of allergenic proteins in a particular species is an important step for the development of more accurate allergy tests and for the definition of more effective management of patients. Currently, studies regarding the allergens in bivalve species are lacking. Materials and methods: This minireview is an overview of local scientific articles on identification of bivalve allergens by allergenomics methods including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE), immunoblotting and mass-spectrometry analysis. Results: Tropomyosin has been the only well recognized allergen in a wide range of bivalve species. In addition to tropomyosin, our local study resulted in discovery of three novel bivalve major allergens; arginine kinase (40 kDa), actin (42 kDa) and troponin C (18 kDa). Conclusion: This minireview will provide a better understanding of each allergen derived from local bivalves which subsequently will help in the understanding of the specific immunoglobulin E(IgE)-mediated immune response.

This study aimed to determine the protein profiles and allergenicity of different body parts and genders of Scylla paramamosain. Raw extracts of mix and six individual body parts were prepared from the respective crab flesh and then analyzed by SDS-PAGE and immunoblotting using sera from 10 crab-allergic patients. The mix body part extract contains 24 protein fractions, while all individual body parts have almost similar protein profiles with 16 to 21 protein bands between 15 to >250 kDa. Various IgE-binding patterns were detected in immunoblotting of mix and individual body parts. Generally, the female leg was identified as the most allergenic part, while the female abdomen was demonstrated as the least allergenic part based on the number and intensity of IgE-binding proteins. Five major allergens at 95, 50, 42, 38 and 36 kDa were identified as potential major allergens. As a conclusion, this study indicated that all body parts of S. paramamosain have numerous and various allergenic proteins, contributing to variable allergenicity between the crab body parts. These findings are useful for the advancement of the diagnosis and management of craballergic patients.

Objectives: To identify major allergens of Asian green mussel (Perna viridis) and to investigate the effect of different cooking processes on the allergenicity of this mussel.Methods: Raw, boiled, fried and roasted extracts of mussel were prepared. Protein profiles were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE). Major allergens and allergenicity patterns of all extracts were then determined by immunoblotting with sera from patients with positive skin-prick tests (SPT) to the raw mussel extract. Mass-spectrometry was used to identify the major allergens of this mussel.Results: SDS-PAGE of raw extract showed 21 protein bands. Smaller numbers of protein bands were detected in the boiled, fried and roasted extracts. The 2-DE gel profile of the raw extract further separated the protein bands to similar to 50 protein spots. Immunoblotting of raw extract exhibited 16 IgE-binding proteins with two proteins of 36 and 18 kDa as the major allergens, whereas the cooked extract revealed 2 IgE-binding proteins. 2-DE immunoblotting of raw extract demonstrated 10 IgE reactive spots. Mass-spectrometry analysis led to identification of two important allergens: tropomyosin (36 kDa) and troponin C (18 kDa).Conclusions: The process of cooking resulted in reduced mussel allergenicity by reducing the number of IgEreactive bands. The order of allergenicity of mussel from lowest to highest, was demonstrated to be, raw > boiled > fried approximate to roasted. The two major allergens of Perna viridis were identified as tropomyosin and troponin C.

In Malaysian and certain Asian countries, snail has high market demand and popular to the local people as food. However, snail is also frequently reported as one of the worst food allergens, dominated by severe symptoms such as asthma and anaphylactic shock. Thus, the aims of this study is to determine the allergenicity of two species of edible snails; the local sea snail, Cerithidea obtusa and the freshwater snail Pomacea canaliculata. Snail extracts were prepared from the snail flesh and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine their protein profiling. Allergenic proteins were detected by immunoblotting test using sera from 10 snail-allergic patients. The snails contain 31 to 34 protein fractions between 11 to >250 kDa. The prominent bands were seen at 33, 42, 74 and 250 kDa. Immunoblotting detected 15 and 16 allergenic proteins in C. obtusa and P. canaliculata, respectively. Three protein fractions at 30, 33 and 42 kDa were identified as the major allergens of C. obtusa, while six major allergens at 30, 33, 42, 74, 124 and 218 kDa were detected in P. canaliculata. Various minor allergens were also detected in both snails. This study indicated that numerous proteins of C. obtusa and P. canaliculata were allergenic. Thus, combined allergen extracts of both snails are essential to be included in diagnosis of snail allergy among local allergic patients.

Allergy diagnosis needs to be improved in polysensitized patients due to the existence of possible confounding factors in this type of patients. Component resolved diagnosis (CRD) is a new concept in the investigation of polysensitized patients. The aim of this study was to evaluate if the utilization of ImmunoCAP ISAC improve the diagnosis of the polysensitized allergic rhinitis patients. Skin prick test (SPT) to 58 crude allergen extracts and CRD (ImmunoCAP ISAC) were carried out for 5 polysensitized allergic rhinitis patients. Two patients had a shellfish allergy and avoidance of shellfish was the only way to prevent an allergic reaction. In contrast, although the remaining three patients had low risk for shellfish allergy, but they were the best candidates for immunotherapy using mite extracts. CRD and particularly ImmunoCAP ISAC have proven to be a valuable diagnostic tool in polysensitized patients. ImmunoCAP ISAC helps refine the individual patient's sensitization profile and predict the potential risk of allergic reactions and improve the selection of patients for immunotherapy.

Snail is one of the worst causes of food allergy. Thus, the aim of this study was to identify the major and minor allergens of the local marine snail (Cerithidea obtusa) and subsequently to investigate the impacts of heat treatment on the IgE-binding activity of snail allergens. Proteins from raw and heat-treated snails (boiled, roasted and fried) were extracted and then resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblotting of all extracts were then performed using sera from patients with snail allergy. The results showed that the raw extract contains numerous protein bands between 12 to>250 kDa. Some thermostable proteins, predominantly the 33 and 42 kDa bands, remained detected in all cooked extracts with decreasing intensities from boiled to roasted to fried extracts, while the majority of thermolabile bands denatured after heating. Boiled snail had more protein bands compared to roasted and fried snails. Immunoblotting of raw extract demostrated 19 IgE-binding bands ranging from 15 to 240 kDa. The thermostable bands of 33 and 42 kDa and a thermolabile of 30 kDa band were identified as the major allergens of this snail. The cooked extracts yielded less allergenic bands. The boiled extract yielded approximately 14 IgE-binding bands with some smeared bands at high molecular weight regions. The roasted extract had lesser IgE-binding bands and the majority appeared as smears, while the IgE-reactivity in the fried extract was less visible and appeared as weak smears. This study indicated that both raw and cooked snails played a crucial role in snail allergenicity, as this species of snail contains both thermostable and thermolabile major allergens. The degree of snail allergenicity was revealed in the order: raw> boiled > roasted> fried. Thus, the results would facilitate in the development of effective diagnosis and management strategies of snail allergy in this country

Shellfish has been recognized as one of the leading causes of food allergy in both adults and children in Asia Pacific region. In Malaysia, black tiger prawn (Penaeus monodon) is among the most widely consumed species. Our previous studies have successfully identified several major allergens including a thermostable protein of 36 kDa. Thus the aim of this study was to identify the 36 kDa major allergen by an allergenomic approach. Protein extracts of raw prown were prepared and resolved by 2-dimensional electrophoresis (2-DE). Immunoblotting was then performed using sera from patients with prawn allergy. Selected spot from 2-DE was then excised, digested and analyzed by mass spectrometry. The 2-DE profile of the extract revealed approximately 100 protein spots between pH of ~4 to 10 and the size range between (<10 to 250 kDa). The 2-DE immunoblotting has detected numerous IgE-binding spots at 36 kDa. Mass spectrometry analysis of the major IgE-binding spot (spot 1a) has identified the 36 kDa spot as tropomyosin. Our findings indicated that tropomyosin play a major role in allergic reaction to black tiger prawn among local patients with prawn allergy, and should be included in diagnostics and therapeutic strategies of this allergy.

Crab meat is widely consumed in several countries around the world. However, when consumed, crab meats are frequent cause of allergic reactions throughout the world. Scylla serrata is among the most common mud crab in Malaysia. In a previous study two major allergens of mud crab at 36 and 41 kDa was identified. Thus, the aim of this study is to further identify these major allergens by a proteomic approach. Protein extract was prepared and resolved by 2-dimensional electrophoresis (2-DE). Immunoblotting was then performed using reactive sera from patients with crab allergy. Major allergenic spots were then excised from the 2-DE gel and analysed by mass spectrometry. The 2-DE profile of the extract revealed approximately >100 protein spots between pH of 4.00 to 8.00. Mass spectrometry analysis has identified the 36 and 41 kDa proteins as tropomyosin and arginine kinase, respectively. Our findings indicated that tropomyosin and arginine kinase play a major role in allergic reaction to mud crab meat among local patients with crab meat allergy, and should be included in diagnostics and therapeutic strategies of this allergy.

Many types of shellfish including oysters are sometime cooked before ingestion and it has been demonstrated that cooking may affect the allergenicity of food. Therefore, the aim of our present study is to identify major and minor allergens of tropical oyster (Crassostrea belcheri) and to investigate the effect of different cooking processing on the allergenicity of this oyster.

Objectives. To identify the major allergenic proteins of clam(Paphia textile) and to investigate the effect of different cooking methods on the allergenicity of these identified proteins. Methods. Clam protein extracts were separated by denaturing polyacrylamide gel electrophoresis. IgE reactive proteins were then analyzed by immunoblotting with sera from patients with positive skin prick tests (SPT) to the raw clam extract. Mass spectrometry was used to identify the major allergenic proteins of this clam. Results. Raw extract showed 12 protein bands (18-150 kDa). In contrast, fewer protein bands were seen in the boiled extract; those ranging from 40 to 150 kDa were denatured. The protein profiles were similarly altered by frying or roasting. The immunoblots of raw and boiled extracts yielded 10 and 2 IgE-binding proteins, respectively. The fried and roasted extracts showed only a single IgE-binding protein at 37 kDa. Mass spectrometry analysis of the 37 and 42 kDa major allergens indicated that these spots were tropomyosin and actin, respectively. Conclusion. The two major allergens of Paphia textile were identified as the thermostable tropomyosin and a new thermolabile allergen actin.