title: Production of in-vitro refolded and highly antigenic SAG1 for development of a sensitive and specific Toxoplasma IgG ELISA.
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.
authors.affiliation: Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran; Recombinant Protein Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, Iran.
authors.forename: Mojgan
authors.initials: M
authors.lastname: Allahyari
authors.affiliation: Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Reyhaneh
authors.initials: R
authors.lastname: Mohabati
authors.affiliation: Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Jalal
authors.initials: J
authors.lastname: Babaie
authors.affiliation: Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Samira
authors.initials: S
authors.lastname: Amiri
authors.affiliation: Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Zahra Jafari
authors.initials: ZJ
authors.lastname: Siavashani
authors.affiliation: Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mehrak
authors.initials: M
authors.lastname: Zare
authors.affiliation: Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Ghazaleh
authors.initials: G
authors.lastname: Sadeghiani
authors.affiliation: Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. Electronic address: majid.golkar@gmail.com.
authors.forename: Majid
authors.initials: M
authors.lastname: Golkar
mesh_terms: D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000954:Antigens, Surface / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D011247:Pregnancy; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods; D014122:Toxoplasma / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D007074:Immunoglobulin G; D015800:Protozoan Proteins; C047168:SAG1 antigen, Toxoplasma
keywords: Diagnosis; Escherichia coli; SAG1; Toxoplasma gondii; in-vitro refolding