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Toxocariasis is a worldwide zoonotic disease caused by the ascarid nematode Toxocara canis. The most common method available for serodiagnosis of toxocariasis is an enzyme-linked immunosorbent assay (ELISA) test using Toxocara excretory-secretory antigen (TES). The present study describes the development of IgG-ELISA based on antiserum prepared against the recombinant arginine kinase of Toxocara canis. Antiserum was prepared against the purified recombinant arginine kinase (AK) using 6-week-old female Japanese white rabbits. Serum samples were collected from experimentally infected BALB/c and C57BL/6 mice at different time periods. The IgG-ELISA was performed using serum samples from mice (infected/uninfected) and TES antigen with antiserum prepared against the recombinant-AK. The optical density (OD450) was measured at 450 nm using a micro-plate ELISA reader. There were significant differences (P<0.01) in the absorbance between infected and control serum samples. Further, we obtained 100% sensitivity for the serum samples from T. canis-infected mice. Therefore, it is suggested that the recombinant-AK based IgG-ELISA could be applied for immunodiagnosis of human toxocariasis. However, it is necessary to evaluate the specificity of this recombinant antigen with similar geohelminth infections.

Arginine kinase (AK) is a member of a highly conserved family of phosphagen kinases. We determined the cDNA sequence of Toxocara canis AK, cloned it in pMAL plasmid and expressed it in Escherichia coli as a fusion protein with maltose-binding protein. The protein has a theoretical molecular mass of 45,376 Da and an estimated isoelectric point (pI) of 8.38. Alignment of the cDNA-derived amino acid sequence of T. canis AK with other phosphagen kinase sequences showed high amino acid identity with other nematode AKs, and phylogenetic analysis placed it as a distinct branch within a nematode AK cluster. Analysis of the N-terminus sequence of T. canis AK revealed the presence of a signal targeting peptide presumably targeting this protein to cytosol or endoplasmic reticulum (ER). T. canis AK showed high activity for l-arginine. The kinetic constants (K(m) = 0.12 mM, K(cat) = 29.18, and K(d) = 0.23 mM) and V(max) (43.76 micromolPi/min/mg protein) of T. canis recombinant-AK were determined for the forward reaction. It also exhibited a synergism for substrate binding (K(d)(Arg)/K(m)(Arg)=1.96). Comparison of K(cat)/K(m)(Arg) values in various arginine kinases indicates that T. canis AK has a high catalytic efficiency (248.19s(-1)mM(-1)). The present study contains the first description of arginine kinase in a zoonotic nematode. The determination of T. canis AK and its phosphagen biosynthetic pathway, which is completely different from those in mammalian host tissues, suggests this enzyme as a possible novel chemotherapy target for VLM syndrome in humans.