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Production of in-vitro refolded and highly antigenic SAG1 for development of a sensitive and specific Toxoplasma IgG ELISA.

Journal of immunological methods (2014-12-02)
Mojgan Allahyari, Reyhaneh Mohabati, Jalal Babaie, Samira Amiri, Zahra Jafari Siavashani, Mehrak Zare, Ghazaleh Sadeghiani, Majid Golkar
ABSTRACT

Recombinant antigens are increasingly applied to replace native antigens in serological tests. Surface antigen 1 (SAG1) is a highly immunogenic antigen and probably represents the most explored and used antigen of Toxoplasma gondii for development of serological test kits. The presence of six disulfide bridges in its structure makes SAG1 a highly conformational protein. In fact, antigenicity of SAG1 is greatly dependent on proper disulfide bonding and folding. In-vitro refolding of SAG1 inclusion bodies, produced in Escherichia coli, was reported to result in soluble and antigenic protein. We produced SAG1 in E. coli and highly purified it by a single denaturing immobilized metal affinity chromatography. Refolding of denatured SAG1 was performed by (a) dialysis in the presence of reduced/oxidized glutathione, (b) drop-wise dilution and (c) drop-wise dilution in the presence of CuSo4. Refolding in the presence of oxido-shuffling reagent was much more efficient in producing presumably correctly-folded and highly antigenic SAG1 as demonstrated by non-reducing SDS-gel electrophoresis, ELISA, Western blotting and reversed-phase HPLC. An IgG ELISA developed using SAG1 refolded in the presence of oxido-shuffling reagent displayed high sensitivity and specificity for detection of Toxoplasma IgG antibodies in pregnant women.

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