title: Design and characterization of a recombinant colorimetric SAG1-alkaline phosphatase conjugate to detect specific antibody responses against Toxoplasma gondii.
abstract: The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to detect specific antibody responses against Toxoplasma gondii in a simple, rapid and highly sensitive reagent. The surface T. gondii SAG1 protein is an important immunodominant target, which provides a great interest as a diagnostic antigen. To further exploit its immunodetection capacity, in the present study, the full length sag1 gene was inserted into the pLIP6 prokaryotic expression vector so as to produce a SAG1 antigen genetically fused to the bacterial alkaline phosphatase (AP). After expression optimization, the recombinant fusion protein folded correctly in soluble form in the periplasmic space and preserved both the AP enzymatic activity and the SAG1 immunoreactivity. Subsequently, direct-ELISA and dot-blot immunoassays were designed, using crude preparation SAG1-AP conjugate, to explore its value in serodiagnosis of human toxoplasmosis. We demonstrate that the recombinant SAG1-AP can detect specific T. gondii antibodies in one-step procedure and can successfully discriminate between T. gondii immune and non-immune patients, in agreement with the standard gold test. In conclusion, the present study shows that the genetic fusion protein provides a new tool for one-step T. gondii immunodetection, which was easily, quickly and reproducibly produced as homogeneous bi-functional reagent. Thus, this recombinant immunoconjugate is a promising marker for Toxoplasma serodiagnosis, requiring further evaluation on a larger series and could provide the basis for direct antibody capture enzyme-immunoassay for specific immunoglobulin M and G detection.
authors.affiliation: Laboratoire de Parasitologie Médicale, Biotechnologies et Biomolécules, LR11-IPT06, Institut Pasteur de Tunis, 13 Place Pasteur-BP74, 1002 Tunis-Belvédère, Tunisia.
authors.forename: Nouha
authors.initials: N
authors.lastname: Chahed Bel-Ochi
authors.forename: Aïda
authors.initials: A
authors.lastname: Bouratbine
authors.forename: Mohamed
authors.initials: M
authors.lastname: Mousli
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000276:immunology*; D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D014122:Toxoplasma / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C047168:SAG1 antigen, Toxoplasma; D000469:Alkaline Phosphatase
keywords: 5-bromo-4-choro-1-indolylphosphate; AP; Alkaline phosphatase; BCIP; ELISA; Fusion protein; IPTG; Immunoenzymatic conjugates; NBT; PBS; SAG1; SAG1 antigen; SDS-PAGE; Serodiagnosis; T. gondii; Toxoplasma gondii; alkaline phosphatase; enzyme-linked immunosorbent assay; isopropyl-β-d-thiogalactoside; nitro-blue tetrazolium; phosphate-buffered saline; sodium dodecyl sulfate poly-acrylamide gel electrophoresis; surface antigen-1