Enzyme-linked immunosorbent assay using recombinant SAG1 antigen to detect Toxoplasma gondii-specific immunoglobulin G antibodies in human sera and saliva

Abstract

Serologic detection of Toxoplasma gondii IgG antibodies is widely accepted as a means to determine immune status and susceptibility to Toxoplasma infection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinant T. gondii SAG1 antigen (rSAG1) to assess its diagnostic performance in Toxoplasma IgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.

Fig 1

Expression and purification analysis. SDS-PAGE analysis of bacterial lysis extract and Coomassie blue staining of rSAG1 antigen (A) and Western blot treated with antipolyhistidine-peroxidase conjugate antibody (B) or a human reference serum sample (C). Numbers correspond to the noninduced cell culture (lane 1), the crude solubilized preparation with 8 M guanidine-HCl (lanes 2), and the purified solubilized preparation under denaturing conditions (6 M urea) (lanes 3). Molecular mass (MW) markers in kDa are indicated on the left.

Fig 2

rSAG1 ELISA results according to the toxoplasmosis immune status of pregnant women. (A) Serum samples; markers correspond to mean ODs ± 2 SD. The dotted lines refer to OD values obtained by the rSAG1 serum-based ELISA for the Platelia kit-positive calibrators (6, 60, and 240 IU). (B) Saliva samples; 0 and 1 refer to nonimmune and immune toxoplasmosis status, respectively.

Fig 3

Receiver operating characteristic (ROC) curve for the rSAG1 saliva-based ELISA applied to positive versus negative individuals identified by the rSAG1 serum-based ELISA.