Enzyme-Linked Immunosorbent Assays Using the Recombinant Dense Granule Antigens GRA6 and GRA1 of Toxoplasma gondii for Detection of Immunoglobulin G Antibodies

Abstract

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 antigenbased test for serodiagnosis of toxoplasmosis.Diagnosis of Toxoplasma gondii infections is of great medical importance for humans, especially pregnant women and immunosuppressed patients. During pregnancy, primary infection of women is often associated with fetal infection, which can lead to abortion or severe neonatal malformations. In immunocompromised adults (e.g., AIDS patients), toxoplasmosis (acute or, most often, reactivation of chronic infection) frequently causes a life-threatening encephalitis (for a review, see reference 12).Among the available diagnostic tests, serology is commonly used. Despite the fact that serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. At present, the detection of specific antibodies based on the recognition of crude Toxoplasma antigens requires mass production of the parasite either from peritoneal fluids of infected mice or from tissue cultures. The use of an Escherichia coli recombinant antigen(s) would be greatly beneficial in improving standardization of the tests and reducing their production cost.Enzyme-linked immunosorbent assay (ELISA) tests using Toxoplasma recombinant antigens have already been reported (4,9,10,11,14,17), but compared to the current serological tests, none of these recombinant antigens has allowed detection of all serologically positive individuals. It has emerged from these studies that the use of two or several recombinant antigens could be necessary to improve the sensitivity of these ELISA tests. Our previous studies on the secreted dense granule (GRA) antigens have shown that the recombinant forms of GRA1 (1), GRA2 (8), and GRA6 (this paper) are recognized by immune human sera. Here we report expression of both the GRA1 and GRA6 proteins in fusion with gl...

Help in the Choice of Automated or Semiautomated Immunoassays for Serological Diagnosis of Toxoplasmosis: Evaluation of Nine Immunoassays by the French National Reference Center for Toxoplasmosis

“…Recent studies revealed that choosing recombinant chimeric multiepitope antigens (54,55) was instrumental in improving the performance of commercialized enzyme immunoassay kits in detecting T. gondii-specific antibodies, using either soluble antigens from crude extracts or recombinant antigens (56)(57)(58). The selection of the reagent that allows for both IgG and IgM detection should take into account the assay's performance under both routine conditions and specified clinical settings (such as high sensitivity for IgM in pregnancy and for IgG in immunosuppressed patients).…”

Toxoplasma gondii Recombinant Antigens as Tools for Serodiagnosis of Human Toxoplasmosis: Current Status of Studies

“…Taken together, these data demonstrated the potential of using two or three complementary recombinant antigens in order to reach a sensitivity comparable to that obtained with a crude antigen preparation. In 2000, Lecordier et al (42) showed that the sensitivity of IgG ELISAs with the single recombinant antigens GRA1 and GRA6-Nt was low (68% and 96%, respectively). They also found that comparison of the optical density values for each serum sample revealed that GRA1 may complement GRA6-Nt to reach an overall sensitivity of 98%.…”

“…Many of these recombinant antigens have been used for the detection of T. gondii-specific antibodies in human serum samples (38). Furthermore, several previous studies have found that recombinant antigens improve the serological diagnosis of toxoplasmosis (15,25,34,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). However, it is not only the improvement of T. gondii diagnosis with the use of recombinant antigens that is very important but, also, improvement in differentiating between the acute and chronic phase of toxoplasmosis.…”

Evaluation of a Recombinant Multiepitope Peptide for Serodiagnosis of Toxoplasma gondii Infection

“…The use of an Escherichia coli recombinant antigen(s) would be greatly beneficial in improving standardization of the tests and reducing their production costs. Thus, recent advances in generating recombinant antigens of T. gondii for IgG and IgM serological tests have been made (10,12,13,17,19,32). However, in contrast to the case for the current serological tests, none of these recombinant antigens has allowed detection of all serologically positive individuals.…”