Identification, production and assessment of twoToxoplasma gondiirecombinant proteins for use in aToxoplasmaIgG avidity assay

Abstract

The IgG avidity assay is an important tool in the management of suspected toxoplasmosis in pregnant women. This study aimed to produce new Toxoplasma gondii recombinant proteins and to assess their usefulness in an IgG avidity assay. Toxoplasma positive and negative serum samples were used, the former were categorized into low (LGA) and high (HGA) IgG avidity samples. Immunoblots were performed on 30 T. gondii cDNA clones to determine the reactivity and IgG avidity to the expressed proteins. Two of the clones were found to have diagnostic potential and were analyzed further; AG12b encoded T. gondii apical complex lysine methyltransferase (AKMT) protein and AG18 encoded T. gondii forkhead-associated (FHA) domain-containing protein. The Histagged recombinant proteins, rAG12b and rAG18, were expressed and tested with LGA and HGA samples using an IgG avidity western blot and ELISA. With the IgG avidity western blot, rAG12b identified 86.4% of LGA and 90.9% of HGA samples, whereas rAG18 identified 81.8% of both LGA and HGA samples. With the IgG avidity ELISA, rAG12b identified 86.4% of both LGA and HGA samples, whereas rAG18 identified 77.3% of LGA and 86.4% of HGA serum samples. This study showed that the recombinant antigens were able to differentiate low avidity and high avidity serum samples, suggesting that they are potential candidates for use in the Toxoplasma IgG avidity assay.

IgG Avidity Test as a Tool for Discrimination between Recent and Distant Toxoplasma gondii Infection—Current Status of Studies

“…However, due to the various limitations of avidity tests based on these antigenic preparation, new diagnostic tools that might improve the performance of avidity tests to distinguish between recent and past phases of toxoplasmosis are tested. In the past 20 years, several studies have reported the use of single recombinant antigens [ 44 , 60 , 61 , 62 , 63 , 64 , 65 , 66 ], mixture of proteins [ 44 , 67 , 68 ] or chimeric proteins [ 69 ] in the determination of IgG avidity ( Table 1 ). All these antigens have been obtained in Escherichia coli expression systems and purified by affinity chromatography.…”

“…Furthermore, Elyasi et al [ 64 ] suggested better performance of avidity ELISA based on GRA6 recombinant antigen than a commercial Euroimmun avidity ELISA for exclusion of a recent infection occurring less than 4 months previously. In 2016, Teh et al [ 66 ] found two novel proteins with diagnostic potential: AG12b encoded T. gondii apical complex lysine methyltransferase (AKMT) protein and AG18 encoded T. gondii forkhead-associated (FHA) domain-containing protein. These are completely new proteins not belonging to antigen families, such as SAG, GRA, MIC, and ROP groups which were tested in the diagnosis of toxoplasmosis.…”