Method for detecting circulating Toxocara canis antigen and its application in human serum samples

Diagnosis of larval migrans (LM) is usually done by immunodiagnostic methods. These methods, however, simply show the presence or absence of antibody but not the active infection of the patients. Therefore, we aimed to establish a diagnostic method for detecting circulating Toxocara canis antigen using a sandwich, ELISA. Monoclonal antibodies (MAb) were produced against the excretory-secretory (ES) antigen of second-stage T canis larvae. Among the MAbs obtained, we selected one MAb (TCMAb12; molecular weight, 30-80 kDa, IgG) for use in the sandwich ELISA. The cross-reactivity of the sandwich-ELISA against thirteen different kinds of parasite antigens were examined. The results revealed that the antibody reacted with T canis ES antigen, T. canis female antigen, and T. canis second-stage larvae antigen, but did not react with any other antigens. From results obtained using an ES antigen concentration standard curve, we confirmed that the detection limit of the sandwich-ELISA was 5 ng/ml, which provides sufficient sensitivity for the diagnosis of toxocariasis (LM). We applied the method to suspected toxocariasis patients and examined the circulating antigen in their sera. We used nine serum samples collected from patients with suspected toxocariasis based on both their clinical symptoms and high antibody titers. Overall, five sera showed antigen-positive reactions, while the remaining four were negative. These results indicated that about 44.0% of the antibody-positive patients were antigen-negative, not ongoing active infection. The results obtained using this technique would provide us for understanding toxocariasis.