Identification of wheat sensitization using an in-house wheat extract in Coca-10% alcohol solution in children with wheat anaphylaxis.

BACKGROUND: Identification of wheat sensitization by a skin prick test (SPT) is essential for children with wheat-induced anaphylaxis, since oral food challenge can cause serious adverse effects. Wheat allergens are both water/salt and alcohol soluble. The preparation of wheat extract for SPT containing both water/salt and alcohol soluble allergen is needed. OBJECTIVE: To determine if a wheat extract using Coca's solution containing 10% alcohol (Coca-10% EtOH), prepared in-house, contians both water/salt and alcohol soluble allergens. METHODS: Serum of children with a history of anaphylaxis after wheat ingestion was used. Wheat flour was extracted in Coca-10% alcohol solution. An SPT with both commercial and in-house wheat extracts was performed as well as specific IgE (sIgE) for wheat and omega-5 gliadin. Direct and IgE inhibition immunoblots were performed to determine serum sIgE levels against water/salt as well as alcohol soluble (gliadins and glutenins) allergens in the extracts. RESULTS: Six children with history of wheat anaphylaxis had positive SPT to both commercial and in-house extracts. They also had different levels of sIgE against wheat and omega-5 gliadin allergens. The results of direct immunoblotting showed all tested sera had sIgE bound to ~35 kDa wheat protein. Further IgE inhibition immunoblotting identified the ~35 kDa wheat protein as gliadin but not gluten allergen. CONCLUSION: The in-house prepared Coca-10% EtOH solution could extract both water/salt and alcohol soluble allergens. The ~35 kDa gliadin appears to be a major wheat allergen among tested individuals.

Cloning and characterization of recombinant tropomyosin of giant freshwater shrimp M. rosenbergii to determine major allergens causing allergic reactions among shrimp-allergic children.

BACKGROUND: Seawater and freshwater shrimp are some of the most common causes of food allergy among children in Thailand. Tropomyosin has been reported as a major allergen for shrimp allergic populations around the world. Despite a high number of shrimp-allergic Thai children, however, it is unknown whether shrimp tropomyosin is a major cause of allergic reactions. OBJECTIVES: To clone and characterize tropomyosin of giant freshwater shrimp Macrobrachium rosenbergii (Mr) and determine whether this tropomyosin is a major cross-reactive allergen for Thai children with shrimp allergy. METHODS: Recombinant shrimp Mr tropomyosin (Mac r1.0101) was expressed in yeast Pichia pastoris. Secondary structure composition of purified recombinant Mac r1.0101 was determined by Circular Dichroism. IgE reactivity was examined by immunoblot, direct binding ELISA and inhibition of IgE ELISA using serum from shrimp-allergic children. RESULTS AND CONCLUSION: The amino acid sequence of Mac r1.0101 showed 2 polymorphic amino acids (F44 and S45) indicating a variant of tropomyosin. Purified recombinant Mac r1.0101 obtained a nature-like α-helix structure which can be bound by serum-specific IgE. The binding affinity of serum-specific IgE to Mac r1.0101 based on the IC50 value was ~1.8 ng/ml. Ten of 13 shrimp-allergic Thai children had serum-specific IgE against Mac r1.0101, but at different levels. Results of the inhibition of specific IgE using Mac r1.0101 showed that 7 of the tested serum samples also had specific IgE against other shrimp allergens in addition to IgE against Mac r1.0101. Tropomyosin therefore appears to be a major cross-reactive allergen for Thai children who are allergic to both seawater and giant freshwater shrimp.