Identification Of Potential Toxoplasma Gondii Cdna Phage Clones And Production Of The Corresponding Recombinant Proteins In The Development Of Igg Avidity Assay For Serodiagnosis Of Toxoplasmosis
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Date
2016-07
Authors
Teh, Ai Ying
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Abstract
Toxoplasmosis is a worldwide infection caused by Toxoplasma gondii. Acute (primary) infection in pregnant women can lead to congenital toxoplasmosis which causes neonatal malformation, neurological damage, eye lesions or fetal death. Therefore, assessment of the stage (chronic or acute) of toxoplasmosis in pregnant women is crucial for appropriate patient management. Serodiagnosis remains the most common approach for laboratory diagnosis of Toxoplasma infection as well as to assess the stage of the infection. Determination of IgG avidity has been proven to be useful in differentiating acute from chronic Toxoplasma infection. The IgG avidity is low at the beginning of the infection and gradually increases over time. High IgG avidity confirms chronic infection while low IgG avidity indicates a probable recent infection. Most of the commercially available Toxoplasma serological assays uses native antigen isolated from in-vivo or in-vitro culture of T. gondii, and they show variability in performance. The recombinantly produced T. gondii antigens are attractive alternatives in the development of an improved IgG avidity assay. Therefore, the present study was conducted to address this need. In this study, 30 T. gondii cDNA phage clones were immunoscreened using serum samples negative for Toxoplasma-specific IgG and IgM (IgG-, IgM-), and the results showed 9 clones with non-reactivity of ≥ 70%. These clones were then tested with serum samples positive for Toxoplasma-specific IgG and IgM (IgG+, IgM+) and showed 7
clones with the reactivity of ≥ 70%. They were further tested with IgG avidity immunoscreening using low IgG avidity (LGA) and high IgG avidity (HGA) serum samples. Two cDNA clones with the highest performance were identified and sequenced. Clone AG12b encoded T. gondii apical complex lysine methyltransferase (AKMT) protein while AG18 encoded T. gondii forkhead-associated (FHA) domain-containing protein. The DNA sequences were cloned into pET32 expression vector; and the His-tagged recombinant proteins rAG12b and rAG18 were expressed and evaluated with IgG avidity western blot and ELISA. With IgG avidity western blot, rAG12b identified 86.4% LGA (n=22) and 90.9% HGA serum samples (n=22); while rAG18 identified 81.8% of each LGA and HGA serum group. With IgG avidity ELISA, rAG12b identified 86.4% of each LGA (n=22) and HGA (n=22) serum group; while rAG18 identified 77.3% LGA and 86.4% HGA serum samples. In general, these results were either better or comparable to the reported IgG avidity assays. In conclusion, this study has identified two T. gondii cDNA phage clones with diagnostic potential for use in IgG avidity assay and successfully expressed and purified the corresponding recombinant proteins as rAG12b and rAG18. The good diagnostic potential demonstrated by the recombinant proteins, in particular, rAG12b, make them potentially useful in the development Toxoplasma IgG avidity assay for serodiagnosis of toxoplasmosis.
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Toxoplasmosis is a worldwide infection , caused by Toxoplasma gondii.