Enzyme-linked immunosorbent assays using the recombinant dense granule antigens GRA6 and GRA1 of Toxoplasma gondii for detection of immunoglobulin G antibodies.

The potential of the dense granule antigens GRA1 and GRA6 of Toxoplasma gondii to be used as diagnosis reagents in a recombinant form was evaluated. Both proteins were expressed in Escherichia coli as glutathione-S-transferase (GST) fusions. The GST-GRA1 fusion comprises the entire GRA1 sequence devoid of its N-terminal signal peptide. Separate expression of the two N- and C-terminal hydrophilic regions of GRA6 showed that only the N-terminal hydrophilic part of the protein was recognized by a pool of positive human sera in an immunoblot. One hundred T. gondii-positive and 98 negative human sera were tested in two separate immunoglobulin G (IgG)-direct enzyme-linked immunosorbent assays (ELISAs) using either GST-GRA1 or GST-GRA6-Nt recombinant protein. Whereas the sensitivity of the GST-GRA1 IgG ELISA was low (68%), the GST-GRA6-Nt IgG ELISA reached a sensitivity of 96%. The reactivity to GRA6-Nt was shown to be high even with human sera of low IgG titers. In addition, comparison of the optical density values for each serum revealed that GRA1 may complement GRA6-Nt to reach an overall sensitivity of 98%. Therefore, the GST-GRA6-Nt ELISA could be used together with another antigen like GRA1 for the development of a recombinant antigen-based test for serodiagnosis of toxoplasmosis.

Similarities between the primary structures of two distinct major surface proteins of Toxoplasma gondii.

Toxoplasma gondii possesses a 43-kDa surface protein (SAG3) that is expressed by all invasive stages. We have cloned and sequenced cDNAs encoding SAG3, with the longest one encoding a primary product of 385 amino acid residues. The deduced amino acid sequence contains a putative NH2-terminal signal sequence, as well as a glycosylphosphatidylinositol anchor attachment site. It is characterized by 12 cysteine residues whose distribution suggests a tandem duplication of a single ancestral motif containing 6 cysteine residues. Although no DNA sequence analogies were found, comparative amino acid sequence analysis detected a resemblance to SAG1, which is the major surface antigen specifically expressed by the proliferative tachyzoite stage. Despite a low degree of identity between the two amino acid sequences (24%), the conservative distribution of the cysteine and tryptophan residues, as well as of repeated motifs, together with oligopeptide identities suggest similar folding and possibly similar function for both proteins.

Amino acid sequence requirements for the epitope recognized by a monoclonal antibody reacting with the secreted antigen GP28.5 of Toxoplasma gondii.

We have characterized in detail, the epitope of the secreted antigen GP28.5 recognized by the mouse monoclonal antibody TG 17-179 using synthetic peptides and a truncated recombinant fusion protein. The screening of a T. gondii expression library with TG17-179 mAb led to the isolation of cDNAs clones, all encoding for the C-terminal region of GP28.5 and with one encoding for only the five last C-terminal residues. Competitive ELISA with longer peptides revealed that the immunoreactivity was retained for peptides of eight residues or longer, and lost when the peptide was reduced to the six last C-terminal residues or less. Experiments with the octapeptide lacking the carboxy-terminal glutamine residue showed it to be 64-fold less active. Moreover, neither addition of residues in the carboxy-end nor substitution of COOH function changed the immunoreactivity of the epitope. Competition experiments between TG17-179 mAb and sera from infected individuals demonstrates that the epitope defined by a mouse monoclonal probe is also a major epitope for human polyclonal antibodies. These results describe the sequence requirements within a probably linear epitope and give rise to some general question concerning experimental test for epitope mapping.