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 CX(17-25)CX(9-22)CX(8)CX(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.
Rodríguez
E
E
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
Anadón
A M
AM
García-Bodas
E
E
Romarís
F
F
Iglesias
R
R
Gárate
T
T
Ubeira
F M
FM
D000485:Allergens / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017129:Anisakiasis / Q000175:diagnosis* / Q000276:immunology; D017163:Anisakis / Q000276:immunology*; D000909:Antibodies, Helminth / Q000097:blood; D000947:Antigens, Helminth / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D015723:Gene Library; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data
D000485:Allergens; D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D000939:Epitopes; D015801:Helminth Proteins