A phage display system for the identification of novel Anisakis simplex antigens

Anisakis simplex has been recognized as an important cause of disease in man and as a foodborne allergen source. Actually, this food-borne was recently identified as an emerging food safety risk including allergenic symptoms. This parasite contains a large variety of allergenic proteins enforcing the necessity to detect new allergens. Commonly, these efforts have been focused on the developing of cDNA libraries, where virtually all expressed mRNAs are present, by using immunoreactive patient serum or polyclonal antibodies. Phage display system is an alternative strategy which permits the physical binding of the genotype with the phenotype, since the products are expressed by the phage on its surface, thereby allowing more efficient selection. In this work we have constructed two libraries in the pJuFo phage, obtaining a primary titer of around 103 cfu/ml and an amplified titer of the order of 1013 cfu/ml whereas the insert sizes varied from 0.35 to 1.2kb. Both libraries were subsequently analyzed by enrichment with polyclonal antibodies to an A. simplex extract and immunoreactive sera from patients with a clinical history of allergy to this parasite. Finally, 30 clones were scrutinized detecting several Anisakis candidate antigens. Actually, one protein, belongs to the fructose-1,6-bisphosphatase family, was found in 34% of scrutinized clones revealing as a promising novel A. simplex allergen. Phage display technology has to date not yet been applied to the identification of new A. simplex allergens, and the present work opens up new avenues to the understanding of the Anisakis allergenic process.