Novel sequences and epitopes of diagnostic value derived from the Anisakis simplex Ani s 7 major allergen *

Background:  Anisakis simplex allergens may cause severe allergic reactions in infected patients. Human anisakiasis can be specifically diagnosed by detection of immunoglobulin E (IgE) antibodies against O ‐deglycosylated nAni s 7 allergen captured by monoclonal antibody (mAb) UA3 (UA3‐ELISA), although the nature of this important allergen is unknown. The aim of this study was to clone and characterize the Ani s 7 major allergen, and to obtain a recombinant fragment suitable for serodiagnosis. Methods:  An Anisakis cDNA library was screened with mAb UA3 and a cDNA clone (rAni s 7) encoding a 1096‐amino acid fragment of Ani s 7 (GenBank: EF158010 ) was identified. Bioinformatic tools and immunological and biochemical techniques were used to characterize the allergen obtained. Results:  The rAni s 7 fragment comprised 19 repeats of a novel C X 17−25 C X 9−22 C X 8 C X 6 tandem repeat motif not seen in any previously reported protein sequence. An internal 435 Met– 713 Arg fragment of the rAni s 7 (t‐Ani s 7) was expressed in Escherichia coli and evaluated for serodiagnostic utility. Indirect enzyme‐linked immunosorbent assay (ELISA) with t‐Ani s 7 identified as positive the same 60 sera as UA3‐ELISA. The sequence MCQCVQKYGTEFCKKRLA from rAni s 7 was identified as the epitope recognized by mAb UA3, and is the target for over 60% of human IgE antibodies that react with O ‐deglycosylated nAni s 7. Conclusions:  In addition to their clear value for serodiagnosis of human anisakiasis, the nature of the novel sequences and epitopes identified in the Ani s 7 allergen are of interest for a better understanding of the mechanisms operating in Anisakis ‐induced allergy.