Development of a highly specific recombinant Toxocara canis second-stage larva excretory-secretory antigen for immunodiagnosis of human toxocariasis

Abstract

The specificity of the recombinant Toxocara canis antigen developed for the immunodiagnosis of human toxocariasis was compared with that of the excretory-secretory antigen from T. canis second-stage larvae (TES) by enzyme-linked immunosorbent assay. A total of 153 human serum samples from patients infected with 20 different helminths, including 11 cases of toxocariasis, were examined. No false-negative reactions were observed for the toxocariasis cases. When the TES was used at concentrations of 0.5 and 0.125 microg/ml, cross-reactions were observed in 79 (55.6%) and 61 (43.0%) of 142 cases, respectively. In contrast, when the recombinant antigen was tested at a concentration of 0.5 microg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a concentration of 0.125 microg/ml, however, the cross-reaction rate decreased sharply to only 2.1%, corresponding to 3 of 142 cases. The cross-reactions occurred with one case each of gnathostomiasis, paragonimiasis with Paragonimus miyazakii, and spirometriasis, in which high antibody titers were detected. In addition, the recombinant antigen showed negative reactions with serum samples from patients infected with Ascaris and hookworms, which are the most common parasites in the world. These findings are also supported by experiments with animals infected with Ascaris and hookworm. From these results, the recombinant antigen is highly specific for toxocariasis and may provide more reliable diagnostic results than other methods.

FIG. 1

Reactivities in ELISA of TES (○) and the recombinant antigen (●) against serum samples from 40 healthy individuals. Each antigen was sensitized at concentrations of 0.5 μg/ml (A) and 0.125 μg/ml (B). Bars are the cutoff points calculated on the basis of three times the OD value for the negative pooled serum. Tc, one patient with toxocariasis as a positive control; NC, individual healthy person.

FIG. 2

Comparison of reactivities in ELISA of TES (○) and the recombinant antigen (●) against serum samples from patients infected with different helminths. For cases of T. canis infection, circles and triangles indicate visceral and ocular toxocariasis, respectively. The concentration of antigens tested was 0.5 μg/ml. Bars denote cutoff points. Abbreviations: Tn, toxocariasis with T. canis; Tc, toxocariasis with T. cati; Bm, brugiasis (B. malayi); Di, dirofilariasis (D. immitis); L1, loiasis; Mp, mansonelliasis (Mansonella perstans); TE, tropical eosinophilia; Gd, gnathostomiasis; Cp, intestinal capillariasis; Ac, angiostrongylosis (Angiostrongylus cantonensis); An, anisakiasis; Al, ascariasis; Ad, ancylostomiasis; Sj, schistosomiasis japonica; Pm, paragonimiasis (P. miyazakii); Pw, paragonimiasis (P. westermani); Fh, fascioliasis; Sp, sparganosis; Sm, spirometriasis; Ec, echinococciasis; Dn, diphyllobothriasis; NC, pooled negative serum.