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Identification of tropomyosin as major allergen of white squid (Loligo edulis) by two-dimensional immunoblotting and mass spectrometry

Yadzir ZH, Misnan R, Murad S

Southeast Asian J. Trop. Med. Public Health, 2012 Jan;43(1):185-91.
PMID: 23082569

IgE-mediated allergic reaction to squid is one of the most frequent molluscan shellfish allergies. Previously, we have detected a 36 kDa protein as the major allergen of Loligo edulis (white squid) by immunoblotting using sera from patients with squid allergy. The aim of this present study was to further identify this major allergen using a proteomics approach. The major allergen was identified by a combination of two-dimensional electrophoresis (2-DE), immunoblotting, mass spectrometry and bioinformatics tools. The 2-DE gel fractionated the cooked white squid proteins to more than 50 different protein spots between 10 to 38 kDa and isoelectric point (pI) from 3.0 to 10.0. A highly reactive protein spot of a molecular mass of 36 kDa and pI of 4.55 was observed in all of the patients' serum samples tested. Mass spectrometry analysis led to identification of this allergen as tropomyosin. This finding can contribute to advancement in component-based diagnosis, management of squid allergic patients, to the development of immunotherapy and to the standardization of allergenic test products as tools in molecular allergology.
Identification of major allergens of wildflower honey

Yadzir ZH, Misnan R, Abdullah N, Arip M, Murad S

Southeast Asian J. Trop. Med. Public Health, 2011 Mar;42(2):370-5.
PMID: 21710860

The aim of this study was to identify the major allergens of wildflower honey in local patients with atopic disease. SDS-PAGE revealed ten protein bands of 25 to 110 kDa, with a heavy cluster in region of 40-75 kDa. Immunoblotting demonstrated seven IgE-binding bands of 39 to 110 kDa. The 60 kDa protein had the highest frequency of IgE-binding (100%) followed by 54 kDa protein (95%), thus identified as the major allergens of wildflowerhoney. Our findings indicate that the allergen extract used for diagnosis of honey allergy contains both the 54 kDa and 60 kDa proteins.
Identification of the major allergens of Charybdis feriatus (red crab) and its cross-reactivity with Portunus pelagicus (blue crab)

Misnan R, Murad S, Yadzir ZH, Abdullah N

Asian Pac J Allergy Immunol, 2012 Dec;30(4):285-93.
PMID: 23393908

Tropomyosin and arginine kinase have been identified as the major allergens in multiple species of crab. Charybdis feriatus is an important commercial crab in this country.
Fulltext Tropomyosin, the major tropical oyster Crassostrea belcheri allergen and effect of cooking on its allergenicity

Yadzir ZH, Misnan R, Bakhtiar F, Abdullah N, Murad S

Allergy Asthma Clin Immunol, 2015;11:30.
PMID: 26504467 DOI: 10.1186/s13223-015-0099-4

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.
Fulltext Tropomyosin and Actin Identified as Major Allergens of the Carpet Clam (Paphia textile) and the Effect of Cooking on Their Allergenicity

Mohamad Yadzir ZH, Misnan R, Bakhtiar F, Abdullah N, Murad S

Biomed Res Int, 2015;2015:254152.
PMID: 26413512 DOI: 10.1155/2015/254152

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.
Identification of Ige-binding proteins of raw and cooked extracts of Loligo edulis (white squid)

Yadzir ZH, Misnan R, Abdullah N, Bakhtiar F, Arip M, Murad S

Southeast Asian J. Trop. Med. Public Health, 2010 May;41(3):653-9.
PMID: 20578555

Allergy to different classes of mollusks, including squid, which are members of the class Cephalopods has been reported. Tropomyosin, a major muscle protein, is the only well-recognized allergen in squid. The aim of this study was to characterize IgE-binding proteins of local Loligo edulis (white squid) consumed in Malaysia. Protein profiles and IgE-binding proteins were detected by sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) and immunoblotting using sera from 23 patients with positive skin prick test to raw squid extract. SDS-PAGE of the raw extract exhibited 21 protein bands (10-170 kDa) but those ranging from 19 to 29 kDa and 41 to 94 kDa were not found in the cooked extract. Immunoblotting of raw extract demonstrated 16 IgE-binding bands, ranging from 13 to 170 kDa. A heat-resistant 36 kDa protein, corresponding to squid tropomyosin, was identified as the major allergen of both extracts. In addition, a 50 kDa heat-sensitive protein was shown to be a major allergen of the raw extract. Our findings indicate that the allergen extract used for diagnosis of squid allergy should contain both the 36 kDa and 50 kDa proteins.
Improved Diagnosis of the Polysensitized Allergic Rhinitis Patients Using Component Resolved Diagnosis Method

Mohamad Yadzir ZH, Bakhtiar F, Misnan R, Abdullah N, Leecyous B, Murad S

Iran J Allergy Asthma Immunol, 2016 Apr;15(2):156-60.
PMID: 27090369

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.
Component-resolved diagnosis (CRD): Is it worth it? frequency and differentiation in rhinitis patients with mite reactivity

Mohamad Yadzir ZH, Misnan R, Abdullah N, Bakhtiar F, Leecyous B, Murad S

Iran J Allergy Asthma Immunol, 2014 Aug;13(4):240-6.
PMID: 24659159

Component-resolved diagnosis (CRD) using microarray technology has recently been introduced with the aim to improve diagnosis of allergy. The aim of this study was to compare performance of this allergen microarray to those of an established extract-based skin prick testing (SPT).45 patients with allergic rhinitis were studied (16 children and 29 adults). SPT to Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis extracts and allergen microarray ImmunoCAP ISAC were carried out for all patients. Forty out of 45 patients demonstrated positive SPT to all mite extracts tested. These 40 patients were considered to be mite-allergic based on the positive SPT results. The remaining 5 patients with negative SPT to any mite extracts were classified as non-mite allergic. Comparatively, based on the microarray results, only 34 mite-allergic patients had detectable serum IgE to at least one of the mite allergen components tested whereas 6 patients with positive SPT to mite extracts showed no detectable IgE reactivity to any of the components tested. One non-mite allergic patient had a positive test- Blo t 5. Der p 10-positive patients also reacted to other cross-reactive tropomyosin from anisakis (Ani s 3) (25%), cockroach (Bla g 7) (50%) and shrimp (Pen m 1) (75%). CRD is a reliable tool for the diagnosis of allergy to mites. Der p 10 might be a useful indicator to identify a subset of mite-allergic patient that have additional sensitization due to cross-reactivity and thus allows selection of patients for immunotherapy.
Impacts of Thermal Treatments on Major and Minor Allergens of Sea Snail, Cerithidea obtusa (Obtuse Horn Shell)

Misnan R, Salahudin Abd Aziz N, Mohamad Yadzir ZH, Bakhtiar F, Abdullah N, Murad S

Iran J Allergy Asthma Immunol, 2016 Aug;15(4):309-316.
PMID: 27921412

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.
Sensitization to Local Seafood Allergens in Adult Patients with Atopic Dermatitis in Malaysia

Lee HL, Tang MM, Bakhtiar MF, Mohamad Yadzir ZH, Johar A

Int Arch Allergy Immunol, 2021;182(2):153-157.
PMID: 32927463 DOI: 10.1159/000510314

BACKGROUND: Seafood is an important source of nutrition in Asia. However, it was believed to cause or aggravate atopic dermatitis (AD).
OBJECTIVES: We aim to determine relevant seafood sensitization among adults with AD and investigate cross-sensitization to aeroallergens.
METHODS: One hundred thirty-two adults with AD who were subjected to skin prick test (SPT) with 7 common local seafood allergens (anchovy, tuna, mackerel, squid, giant freshwater prawn, shrimp, and crab), house dust mites (HDMs), and cockroach were analyzed retrospectively.
RESULTS: The median age of the study subjects was 32 years (range 17-77 years) with a male to female ratio of 1:3. The mean duration of AD was 16 years. Eighty-two patients (62.2%) had other atopic conditions. Using SCORAD, 44.7% had mild, 42.4% moderate, and 12.9% severe disease. Eighty-six patients (65.2%) self-reported to have seafood allergy, with the main symptoms of transient pruritus and erythema within 2 h of ingestion. SPT revealed 51.5% of the patients were sensitized to at least 1 of the 7 seafood allergens. The relevant sensitization rate was 45.1%. Interestingly, 46% of those without a history of seafood allergy developed at least 1 positive reaction in the SPT. Prawn, shrimp, and crab were the 3 most frequently sensitized allergens. Nearly all patients (98.3%) who were sensitized to crustaceans were also sensitized to HDMs and/or cockroach. There was no significant correlation between a positive SPT to seafood with age, age of onset of AD, duration, and severity of AD, and the presence of other atopic diatheses.
CONCLUSION: The relevant sensitization rate of local seafood among adults with AD was 45.1%.
Fulltext Identification of the major allergen of Macrobrachium rosenbergii (giant freshwater prawn)

Yadzir ZH, Misnan R, Abdullah N, Bakhtiar F, Arip M, Murad S

Asian Pac J Trop Biomed, 2012 Jan;2(1):50-4.
PMID: 23569834 DOI: 10.1016/S2221-1691(11)60189-5

OBJECTIVE: To characterize the major allergens of Macrobrachium rosenbergii (giant freshwater prawn).
METHODS: Raw and cooked extracts of the giant freshwater prawn were prepared. The IgE reactivity pattern was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technique with the sera of 20 skin prick test (SPT) positive patients. The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional (2-DE) electrophoresis, mass spectrometry and bioinformatics tools.
RESULTS: SDS-PAGE of the raw extract showed 23 protein bands (15-250 kDa) but those ranging from 40 to 100 kDa were not found in the cooked extract. From immunoblotting experiments, raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins, respectively, with a molecular mass ranging from 15 to 155 kDa. A heat-resistant 36 kDa protein was identified as the major allergen of both extracts. In addition, a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract. The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots. Of these, 10 spots showed specific IgE reactivity with patients' sera. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis led to identification of 2 important allergens, tropomyosin and arginine kinase.
CONCLUSIONS: It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.
Fulltext Identification of major and minor allergens of black tiger prawn (Penaeus monodon) and king prawn (Penaeus latisulcatus)

Sahabudin S, Misnan R, Yadzir ZH, Mohamad J, Abdullah N, Bakhtiar F, et al.

Malays J Med Sci, 2011 Jul;18(3):27-32.
PMID: 22135598 MyJurnal

BACKGROUND: Prawns and shrimp are a frequent cause of seafood allergy mediated by IgE antibodies. Penaeus monodon and Penaeus latisulcatus, commonly known as black tiger prawn and king prawn, respectively, are among the most frequently consumed prawns in Malaysia. The aim of this study was to identify the IgE-binding proteins of these 2 prawn species.
METHODS: Raw and boiled prawn extracts were prepared and then resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). IgE-immunoblotting was then performed using sera from patients with positive skin prick tests to the raw prawn extracts.
RESULTS: SDS-PAGE analysis of the raw extracts of both prawn species revealed 23 protein bands; the boiled extracts yielded fewer protein bands. The bands in the range of 40 to 100 kDa were sensitive to heat and therefore were not found in the boiled extracts. Immunoblot of raw extracts of black tiger prawns and king prawns yielded 14 and 11 IgE-binding proteins, respectively, with molecular weights of between 15 and 200 kDa. Proteins at 36, 42, and 49 kDa were detected as the major allergens in both species of prawns. A protein of 75 kDa was also identified as a major allergen in black tiger prawns. Other potential allergens were also observed at various molecular masses.
CONCLUSION: Proteins of 36, 42, and 49 kDa were identified as the major allergens of both species of prawns. The 36 and 42 kDa proteins are hypothesised to be tropomyosin and arginine kinase, respectively. A high molecular weight protein of 75 kDa was found to be an additional major allergen in black tiger prawns.
KEYWORDS: Penaeus; allergens; allergy and clinical immunology; hypersensitivity; immunoblotting; tropomyosin