Diagnosing human anisakiasis: recombinant Ani s 1 and Ani s 7 allergens versus the UniCAP 100 fluorescence enzyme immunoassay

Abstract

Commercially available serological methods for serodiagnosis of human anisakiasis either are poorly specific or do not include some of the most relevant Anisakis allergens. The use of selected recombinant allergens may improve serodiagnosis. To compare the diagnostic and clinical values of enzyme-linked immunosorbent assay (ELISA) methods based on Ani s 1 and Ani s 7 recombinant allergens and of the UniCAP 100 fluorescence enzyme immunoassay (CAP FEIA) system, we tested sera from 495 allergic and 25 non-food-related allergic patients. The decay in specific IgE antibodies in serum was also investigated in 15 positive patients over a period of 6 to 38 months. Considering sera that tested positive by either Ani s 1 or Ani s 7 ELISA, the CAP FEIA classified 25% of sera as falsely positive, mainly in the group of patients with the lowest levels of anti-Anisakis IgE antibodies, and 1.28% of positive sera as falsely negative. Considering allergens individually, the overall sensitivities of Ani s 7 ELISA and Ani s 1 ELISA were 94% and 61%, respectively. The results also showed that anti-Anisakis IgE antibodies can be detected in serum for longer with Ani s 1 ELISA than with Ani s 7 ELISA and CAP FEIA (P < 0.01). Our findings suggest that ELISA methods with Ani s 7 and Ani s 1 allergens as targets of IgE antibodies are currently the best option for serodiagnosis of human anisakiasis, combining specificity and sensitivity. The different persistence of anti-Ani s 1 and anti-Ani s 7 antibodies in serum may help clinicians to distinguish between recent and old Anisakis infections.

FIG. 1.

(A) SDS-PAGE and WB analysis of Ani s 1 purified protein after oxidative refolding under reducing (lanes 1 to 3) and nonreducing (lanes 4 to 7) conditions. Lane 1, unretained fraction, Coomassie staining; lane 2, retained fraction, Coomassie staining; lane 3, retained fraction revealed by WB with a monoclonal anti-His antibody by a standard procedure; lane 4, retained fraction, Coomassie staining; lane 5, retained fraction revealed by WB with an anti-His antibody as described above; lanes 6 and 7, retained fraction revealed by WB with a positive human serum at 1:4 and 1:25 dilutions, respectively. (B) Chromatogram obtained after size-exclusion chromatography, using a Superose 12 HR column (Pharmacia), of the retained protein fraction of Ani s 1 after purification by Ni-NTA affinity chromatography. Peak 1 corresponds to the monomer fraction. (C) Complete amino acid sequence of Ani s 1 encoded by the pRSET A vector and expressed in BL21(DE3)pLys cells. The specific Anisakis expressed sequence is shown in boldface type. Note that the Ani s 1 sequence differs by one amino acid (Arg to Gln; boxed) from that described by Shimakura et al. (19).

FIG. 2.

Optical density values obtained for the 495 sera investigated in the Ani s 1 ELISA and the Ani s 7 ELISA. Each circle corresponds to an individual serum sample. The sera were grouped into CAP FEIA classes as described in Materials and Methods.

FIG. 3.

(A) Percentages of positive serum samples, as determined by Ani s 1 ELISA (open circles) and Ani s 7 ELISA (closed circles), in CAP FEIA classes 1 to 6, according to the proposed gold standard (see Materials and Methods). The numbers 1 to 6 refer to CAP FEIA classes 1 to 6. (B) Percentage of false-positive sera for each CAP FEIA class, assuming the same gold standard.