Development of double sandwich enzysm linked Immunosorbent assay in the diagnosis of toxocariasis

Abstract

Toxocariasis is an important zoonosis of humans caused by ascarid larvae primarily Toxocara canis from dogs and Toxocara cati from cats. The diagnosis of human toxocariasis currently depends on immunological examinations because it is extremely difficult to detect an infective Toxocara larva (e) in biopsy samples. Excretory - secretory (ES) antigen derived from second-stage larvae of Lcanis maintained in defined medium in vitro has been well established worldwide for the immunodiagnosis of human toxocariasis by enzyme linked immunosorbent assay (ELISA). However, false positives due to cross-reactions with other helminths (commonly found in tropical countries) have been reported for this assay when T.canis ES antigen was used.

Therefore this study was carried out to isolate Lcanis specific ES antigen in order to develop double sandwich ELISA for use in the diagnosis oftoxocariasis in Sri Lanka.

Toxocara canis ES antigen, Ascaris lumbricoides ES antigen and Necator americanus (hook worm) larval antigen were subjected to SDS - PAGE followed by western blotting with hyper - immune sera collected from rabbits in order to identify Lcanis specific protein band. Subsequently , hyper-immune sera in rabbits were produced by injecting the specific protein band. Finally a double sandwich ELISA was developed using T.canis specific antigen. Test was evaluated with 400 T.canisL2 larval ES antigen ELISA positive serum samples. Of the samples tested 10% were negative on the double sandwich ELISA. False positive reactions were not found in patients with parasitologicaly confirmed Ascaris lumbricoides and hook worm infestations.

Thus double sandwich ELISA using T.canis specific antigen is more specific than T.canis L2 larval ES antigen ELISA for diagnosis of human toxocariasis.