Development, optimization, and validation of an in-house Dot-ELISA rapid test based on SAG۱ and GRA۷ proteins for serological detection of Toxoplasma gondii infections

Background and Aim : The aim of the present study was to develop a simple, portable, and rapid assay for serodiagnosis of toxoplasmosis based on recombinant Toxoplasma gondii (T. gondii) SAG۱ (rSAG۱) and GRA۷ (rGRA۷) proteins.Methods : The rSAG۱ and rGRA۷ proteins were expressed in Escherichia coli (E. coli) and purified in a single step by immobilized metal ion affinity chromatography. The immunoreactivity of the recombinant antigens was tested in an in-house IgG and IgM Dot enzyme-linked immunosorbent assay (Dot-ELISA) for potential use in serodiagnosis of T. gondii infection.Results : Results from the comparison of in-house rSAG۱-Dot-ELISA with ELISA for the detection of anti-Toxoplasma IgG and IgM include sensitivity of ۸۳.۷% and ۸۱.۲%, specificity of ۹۰.۲% and ۸۹.۳%, positive predictive values of ۸۵.۹% and ۶۸.۴%, and negative predictive values of ۸۸.۶% and ۹۴.۳%, respectively. Sensitivity of ۶۶.۲%, specificity of ۸۱.۲%, positive predictive values of ۷۱.۶%, and negative predictive values of ۷۷.۱% were concluded from in-house IgG rGRA۷-Dot-ELISA. The sensitivity and specificity of IgM rGRA۷-Dot-ELISA included ۸۷.۵% and ۸۳.۹%, respectively. Sensitivity and specificity of in-house Dot-ELISA for a combination of rSAG۱ and rGRA۷ included ۸۷.۵% and ۹۱.۱% for IgG and IgM, respectively. Sensitivity and specificity of a combination of rSAG۱ and rGRA۷ for the detection of IgM in suspected sera to acute toxoplasmosis were higher than those for the detection of IgG in sera with chronic infections (۹۰.۶% and ۹۲% instead of ۸۶.۲% and ۹۱.۶%, respectively).Conclusion : The highlighted parameters of combined recombinant proteins were more significant than those of single recombinant proteins in in-house Dot-ELISA. These data suggest that the in-house Dot-ELISA based on rSAG۱ and rGRA۷ combination is a promising diagnostic tool with a similar sensitivity to the native antigens of T. gondii, which can be used for the serodiagnosis of toxoplasmosis in fields as well as less equipped laboratories.

Keywords:

Toxoplasma gondii RH strain , in-house Dot-ELISA , rSAG۱ , rGRA۷ , soluble tachyzoite antigen (STAg) , recombinant proteins