title: Expression, purification and in vitro refolding of the recombinant truncated Saposin-like protein 2 antigen for development of diagnosis of human fascioliasis.
abstract: Early diagnosis of fascioliasis is critical in prevention of injury to the liver and bile ducts. Saposin-like protein (FhSAP-2) is probably the most ideal antigen of Fasciola hepatica for development of ELISA kits. SAP-2 has a conserved tertiary structure containing three disulfide bonds and conformational epitopes. Therefore, antigenicity of SAP-2 is greatly depends on disulfide bond formation and proper folding. We produced the recombinant truncated SAP-2 (rtSAP-2) in the SHuffle®T7 and Rosetta strain of Escherichia coli, in soluble and insoluble forms, respectively and purified by immobilized metal affinity chromatography (IMAC). The refolding process of denatured rtSAP-2 was performed using dialysis and dilution methods in the presence of chemical additives, along with reduced/oxidized glutathione (in vitro). Physicochemical studies, including non-reducing gel electrophoresis, Ellman's assay, Western blotting and ELISA showed the most antigenicity and likely correct folding of rtSAP-2, which was obtained by dialysis method. An IgG ELISA test was developed using rtSAP-2 refolded by dialysis and compared with excretory/secretory products of parasite with 52 positive fascioliasis samples, 79 other parasitic samples and 70 negative controls samples. The results exhibited 100% sensitivity and 98% specificity for rtSAP-2, also, 100% and 95.3% for excretory/secretory (E/S) antigen, respectively. In conclusion, it is suggested that rtSAP-2 with the correct folding could be used as a candidate antigen for detection of human fascioliasis.
authors.affiliation: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. Electronic address: ab.mirzadeh85@yahoo.com.
authors.forename: Abolfazl
authors.initials: A
authors.lastname: Mirzadeh
authors.affiliation: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. Electronic address: z_valadkhani@pasteur.ac.ir.
authors.forename: Zarrintaj
authors.initials: Z
authors.lastname: Valadkhani
authors.affiliation: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. Electronic address: asiyeh.yoosefy64@gmail.com.
authors.forename: Asiyeh
authors.initials: A
authors.lastname: Yoosefy
authors.affiliation: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. Electronic address: jalalbabaie@gmail.com.
authors.forename: Jalal
authors.initials: J
authors.lastname: Babaie
authors.affiliation: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. Electronic address: majid.golkar@gmail.com.
authors.forename: Majid
authors.initials: M
authors.lastname: Golkar
authors.affiliation: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. Electronic address: aresmar40@yahoo.com.
authors.forename: Ahmad Reza
authors.initials: AR
authors.lastname: Esmaeili Rastaghi
authors.affiliation: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. Electronic address: ekazemirad@yahoo.com.
authors.forename: Elham
authors.initials: E
authors.lastname: Kazemi-Rad
authors.affiliation: Department of Parasitology, Guilan University of Medical Sciences, Rasht, Iran. Electronic address: k_ashrafi@gums.ac.ir.
authors.forename: Keyhan
authors.initials: K
authors.lastname: Ashrafi
mesh_terms: D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D000947:Antigens, Helminth / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005210:Fasciola hepatica / Q000276:immunology*; D005211:Fascioliasis / Q000175:diagnosis* / Q000469:parasitology; D015801:Helminth Proteins / Q000276:immunology*; D006801:Humans; D017510:Protein Folding; D049231:Saposins / Q000276:immunology*; D012680:Sensitivity and Specificity; D012698:Serologic Tests
chemical_list: D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D015801:Helminth Proteins; D049231:Saposins
keywords: Antigenicity; ELISA; Fasciola hepatica; In vitro refolding; rtSAP-2