Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting.

Toxoplasma gondii trophozoites (RH strain) were cultured in embryonic fibroblasts in order to study the kinetics of production of excretory/secretory antigens, and the results were compared to the production of circulating antigens in an in vivo mouse model. By capture-ELISA, excretory/secretory antigens were first detected on the fourth day of culture whereas circulating antigens were first detected 1 day after infection. Similar concentrations of antigens were detected in both models as evidenced by comparable absorbance values. By immunoblotting, the excretory/secretory antigens were also detected later compared to circulating antigens (day 4 vs day 1). Seven major polypeptides were detected in both antigen preparations, six of them having the same molecular mass (110, 75, 48, 30, 24 and 22 kDa).

Detection of circulating antigens of Toxoplasma gondii in human infection.

Seventy-nine serum specimens from pregnant women and 29 from immunocompromised patients (12 from graft recipients and 17 from patients with acquired immunodeficiency syndrome) were classified into three groups according to their serologic status to Toxoplasma gondii as determined by immunofluorescence and an enzyme-linked immunosorbent assay (ELISA): no antibodies (group 1), acute acquired infection (group 2), and reactivation (group 3). These samples were tested for the presence of circulating antigens (CAg) of T. gondii by capture ELISA and immunoblotting. The presence of CAg was detected by at least one of the two techniques in six of 31 subjects in group 1, 51 of 68 subjects in group 2, and seven of nine subjects in group 3. Of a total of 108 serum specimens, 28 were found to be T. gondii-positive by capture ELISA, 57 by immunoblotting, and 21 by both techniques. Among the nine polypeptides detected by immunoblotting, 38 recognized p14, 17 recognized p8, and 16 recognized p8, and 16 recognized p30. These results demonstrate that the detection of CAg can aid in the diagnosis of infection by T. gondii in humans, especially in immunocompromised patients whose serologic response can be impaired.

Kinetic of circulating antigens by capture ELISA and immunoblotting in murine toxoplasmosis.

Capture ELISA and immunoblotting techniques were applied for the detection of Toxoplasma gondii (T.g) circulating antigens (CAG) in serum specimens of OF-1 mice infected with virulent RH, or cystogenic PGD strains. The capture ELISA assay allowed to detect CAG from the first day of infection to the death of animals with the two T.g strains, although CAG rates were higher when the RH strain was used. Immunoblotting confirmed these findings. Moreover, immunoblotting allowed to identify 7 CAG (MW: 22 kDa to 110 kDa) in serum specimens of mice infected with the RH strain and only 3 (MW: 75 kDa to 110 kDa) in serum specimens of mice infected with the PGD strain. These results are discussed in the light of the evolutive ways of experimental infection by T.g.

Toxoplasma gondii antigens: Analysis by SDS-PAGE and immunoblotting; Relation with circulating antigens.

Cytoplasmic antigenic extracts (CAE) and membranar antigenic extracts (MAE) from Toxoplasma gondii (T.g) purified trophozoites RH strain, were obtained after hypotonic lysis and sonication. Analysis in Polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie blue staining revealed 21 polypeptides (PP) in CAE and 8 in MAE, whereas the immunoblotting technique detected 28 PP in CAE and 23 in MAE. Immunoblotting is then more sensitive than Coomassie blue staining. This finding is particularly marked for MAE and could be explained by the high antigenicity of some PP that have a lower concentration than the threshold sensitivity of SDS-PAGE. In contrast, some PP in sufficient amount to be detected by SDS-PAGE did not show any antigenic activity since not found by immunoblotting. The molecular weight (MW) ranged from 8 to 130 kDa for cytoplasmic PP and from 4 to 110 kDa for membranar PP. Circulating antigens (CAG) from serum specimens of mice infected with the same strain of T.g were analysed by immunoblotting in comparison with CAE and MAE profiles. From the 7 bands obtained with CAG, 4 were shown to correspond to CAE PP, 2 to common CAE and MAE PP and only 1 to MAE PP (MW: 30 kDa).