Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Abstract

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 micro g/mL. The concentration of 250 micro g/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

Potential immunological markers for diagnosis and therapeutic assessment of toxocariasis

“…Our results show some similarities to and discrepancies from reported findings 1,11,16 , although some studies used different immunological approaches for toxocariasis.…”

“…However, there are few follow-up studies in patients after chemotherapy showing changes in their IB patterns, according to antibody isotypes and antigen fractions. For toxocariasis, there have been attempts to find the most suitable antigens or refined antigen epitopes for immunoassays 1,11,16 . There is a need to standardize serodiagnostic criteria and define the efficacy of chemotherapy 23 .…”

In vitro production of Toxocara canis excretory-secretory (TES) antigen

“…Other media were also found to be good to maintain larval culture such as HMEM (de Savigny 1975), Eagle's minimum essential medium (Gupta 1984), Dulbecco's modified Eagles medium (Boyce et al 1988 andMorales et al 2002). However HEPES buffered RPMI 1640 medium containing L glutamine was found to be good for larval culture in terms of better survival of larvae and higher yield of ES products (Badley et al 1987 andRajapakse et al 1992) as observed in the present study.…”

Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis

“…As revealed by western blot, there are two main groups of bands present in the positive cases: one of 24-45 kDa and another group of 66-200 kDa (Magnaval et al 1991, Nunes et al 1997, Morales et al 2002. These studies suggest the HMW bands can cause cross-reactivity and the LMW bands would be more specific for Toxocara.…”