Characterization of allergens secreted by Anisakis simplex parasite: clinical relevance in comparison with somatic allergens

Abstract

Background: Diagnostic methods for the study of allergic reactions to Anisakis simplex (A.s.) based on whole-body extracts of the larva are clearly insufficient. Objectives: To study the allergenicity of the proteins secreted by the parasite. Comparison with somatic antigens and determination of their clinical importance in allergic patients were also addressed. Methods: An excretory/secretory (E/S) extract was produced by culturing third-stage A.s. larvae. It was used to perform immediate skin tests and to determine specific IgE in 10 patients diagnosed with allergy to A.s. Both tests were compared with the results obtained with the whole-body extract (somatic (S)). The molecular weight (MW) of their allergens was determined by immunoblotting, and a single-blind placebo-controlled oral challenge with E/S proteins was performed. Finally, allergens' resistance to gastric pepsin and acid pH was explored. Results: A.s. larvae secreted allergens more potent than those present in the S extract. The skin prick test wheal area produced by E/S molecules and the absorbance obtained in the determination of specific IgE with these allergens (ELISA) were 5.8 times bigger than those obtained with S extract. MW allergens of 72 and 56 kDa in E/S extracts and those of 56, 48 and 43 kDa in S extract were recognized by more than 50% of the patients. Partial cross-reactivity between them was revealed by immunoblotting inhibition studies. Oral challenge with E/S extract (up to 479 microg) was negative in all the patients. Treatment of E/S proteins with gastric pepsin inhibited the binding of the E/S allergens for specific IgE. The acid pH did not affect the overall binding of IgE to E/S extract. It decreased by 15.23% and 19.96% at pH 4 and 2, but the difference was not statistically significant. Conclusion: A.s. secretes allergens more potent than somatic antigens and should be used in the diagnostic procedures. These allergens are inactivated by the pepsin, which supports the theory that live larva is necessary to induce an allergic reaction in most of the patients.