Identification, production and assessment of two Toxoplasma gondii recombinant proteins for use in a Toxoplasma IgG 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 His-tagged 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.

Keywords: ELISA; IgG avidity; Immunoblot; Recombinant proteins; Toxoplasma gondii; Toxoplasmosis; Western blot; cDNA clones.

Figure 1

Immunoblot of AG12b with (A) Group I and (B) Groups II and III serum samples. In 1(A), nitrocellulose membrane pieces AS1–AS5 were incubated with individual serum samples from Group I (IgG-, IgM-) showed non-reactivity; PC was incubated with pooled positive serum as positive control. In 1(B), nitrocellulose membrane pieces RL1–RL5 incubated with individual serum samples from Group II [IgG+, IgM+, low IgG avidity (LGA)] and RH1–RH5 incubated with serum samples from Group III [IgG+, IgM+, high IgG avidity (HGA)] showed good reactivity; NC was membrane piece incubated with pooled negative serum as negative control.

Figure 2

IgG avidity immunoblot of (A) clone AG12b and (B) clone AG18. The decrease in the number of spots on nitrocellulose membrane pieces incubated with HGA serum (RH1, RH2, and RH3) after TBST-6M urea wash was <50% as compared to TBST wash, indicating high IgG avidity reaction. Meanwhile, the number of spots on nitrocellulose membrane pieces incubated with LGA serum samples (RL1, RL2, and RL3) after TBST-6M urea wash was decreased >50% as compared to TBST wash, indicating low IgG avidity reaction.

Figure 3

SDS-PAGE and western blot analysis of purified recombinant proteins (A) rAG12b and (B) rAG18. Lane 1: Coomassie blue-stained SDS-PAGE gel; Lane 2: western blot probed with anti-His-HRP. The molecular mass of rAG12b is ~38 kDa and rAG18 is ~65 kDa. Lane M: molecular weight marker [Precision Plus Protein Unstained Standards (Bio-Rad)].

Figure 4

IgG avidity western blots of (A) rAG12b and (B) rAG18. Lanes 1 and 2 are paired strips incubated with HGA serum sample while lanes 3 and 4 are paired strips incubated with LGA serum sample. Lanes 1 and 3 were treated (washed) with TBST while lanes 2 and 4 were treated with TBST containing 8M urea. When lane 2 was compared to lane 1, the intensity ratio was >0.5, indicating high avidity binding of anti-Toxoplasma IgG to the antigen. When lane 4 was compared to lane 3, the intensity ratio was <0.5, indicating low avidity binding of anti-Toxoplasma IgG to the antigen.

Figure 5

Avidity index scores of IgG avidity ELISA using (A) rAG12b and (B) rAG18. Avidity index at 50%, indicated with a bold dashed line, was used to differentiate acute and chronic serum samples. Of the 22 LGA serum samples tested, rAG12b demonstrated 19 low IgG avidity and 2 high IgG avidity results. Avidity status of one serum sample could not be determined since the OD was lower than the cut-off value. With the same group of serum samples, rAG18 demonstrated 17 low IgG avidity and 3 high IgG avidity results, while the avidity status of 2 serum samples could not be determined. Both recombinant proteins demonstrated 19 high IgG avidity results and 3 low IgG avidity results with the 22 HGA serum samples.