Molecular characterization of a cDNA encoding an excretory–secretory antigen from Toxocara canis second stage larvae and its application to the immunodiagnosis of human toxocariasis

The cDNA encoding an excretory–secretory antigen from the second stage larvae of Toxocara canis has been characterized. Sequence analysis revealed an open reading frame encoding a protein of 226 amino acid residues (Mr=24 398). Sequence database searches showed similarities to regions corresponding to epidermal growth factor-like and lectin-like domains of the core proteins of vertebrate chondroitin sulfate proteoglycans, which are major components of the extracellular matrix. The T. canis core protein was expressed as a fusion protein with thioredoxin A using an Escherichia coli expression system, and then affinity purified on a metal affinity resin in the presence of 8 M urea. When the purified recombinant T. canis protein was used as an antigen, immunoblot analysis revealed the protein specifically reacted with sera from toxocariasis patients. The antigenic protein did not react with sera from patients with Brugia malayi infection, dirofilariasis, or ascariasis. In some cases of anisakiasis, cross-reactions were observed; however, the cross-reacting bands disappeared when anisakiasis sera preabsorbed with Anisakis antigen were used, indicating that the recombinant T. canis protein is very promising for use as an immunodiagnostic antigen for human toxocariasis

Development of a Highly Specific Recombinant Toxocara canis Second-Stage Larva Excretory-Secretory Antigen for Immunodiagnosis of Human Toxocariasis

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 μg/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 μg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a concentration of 0.125 μg/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