Haemoglobin, a new major allergen of Anisakis simplex

Gastro-allergic anisakiasis and Anisakis sensitisation associated chronic urticaria are diseases which differ in their IgE and IgG4 responses against both crude extract and specific allergens. Anisakis and Ascaris are closely related nematodes that usually cause problems with specificity in immunodiagnostics. In this study we measured IgE and IgG4 antibodies against Anisakis simplex sensu lato (s. l.) and Ascaris suum haemoglobins in sera of 21 gastro-allergic anisakiasis and 23 chronic urticaria patients. We used a capture ELISA with the anti-Anisakis haemoglobin monoclonal antibody 4E8g, which also recognises Ascaris haemoglobin. In addition, we determined specific IgE and IgG4 to both nematodes by indirect ELISA and immunoblotting. Anti-A. simplex s. l. haemoglobin IgE and IgG4 levels were higher in gastro-allergic anisakiasis than in chronic urticaria patients (P = 0.002 and 0.026, respectively). Surprisingly, no patient had detectable IgE levels against A. suum haemoglobin. Finally, we carried out an in silico study of the B-cell epitopes of both haemoglobin molecules. Five epitopes were predicted in Anisakis pegreffii and four in A. suum haemoglobin. The epitope propensity values of Anisakis haemoglobin in the equivalent IgE binding region of the allergenic haemoglobin Chi t 1 from Chironomus thummi, were higher those of the Ascaris haemoglobin. In conclusion, we describe A. simplex haemoglobin as a new major allergen (Ani s 13), being recognised by a large number (64.3%) of sensitised patients and up to 80.9% in patients with gastro-allergic anisakiasis. The presence of a specific epitope and the different values of epitope propensity between Anisakis and Ascaris haemoglobin could explain the lack of cross-reactivity between the two molecules. The absence of IgE reactivity to Ascaris haemoglobin in Anisakis patients makes Anisakis haemoglobin (Ani s 13) a potential candidate for developing more specific diagnosis tools.