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Enhancing the detection of Toxoplasma gondii via an anti-SAG1 scFv-alkaline phosphatase immunoconjugate.

ABSTRACT: The purpose of this study was to implement a fluorometric method for enhancing the detection sensitivity of Toxoplasma gondii in biological fluids. To address this challenge, we designed and produced a recombinant immunoconjugate tool based on a single-chain antibody fragment anti-T. gondii SAG1 antigen (scFvSG15) genetically fused to the bacterial alkaline phosphatase (AP) using 4-methyl-umbelliferyl-phosphate as fluorogenic substrate. The anti-SAG1 scFv-AP conjugate was fully bifunctional and was used successfully in different assays including immunoblot, fluorometric ELISA and direct immunofluorescence. The fluorometric immunoassay afforded an extremely low detection limit (1 tachyzoite/well), which was in agreement with the real-time PCR control test. The immunofluorescence imaging has provided captivating visual evidence of T. gondii detection. These results strongly suggest that the recombinant anti-SAG1-AP conjugate generated here might serve as useful and highly sensitive immunoassay probe to direct detect T. gondii in a one-step procedure, opening up new perspectives for diagnosis of toxoplasmosis.

SUBMITTER: Hannachi E

PROVIDER: S-EPMC6630058 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Enhancing the detection of Toxoplasma gondii via an anti-SAG1 scFv-alkaline phosphatase immunoconjugate.

Hannachi Emna E Bouratbine Aïda A Mousli Mohamed M

Biotechnology reports (Amsterdam, Netherlands) 20190705

The purpose of this study was to implement a fluorometric method for enhancing the detection sensitivity of Toxoplasma gondii in biological fluids. To address this challenge, we designed and produced a recombinant immunoconjugate tool based on a single-chain antibody fragment anti-T. gondii SAG1 antigen (scFvSG15) genetically fused to the bacterial alkaline phosphatase (AP) using 4-methyl-umbelliferyl-phosphate as fluorogenic substrate. The anti-SAG1 scFv-AP conjugate was fully bif ...[more]

PMID: 31341791

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Project description:Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan parasite T. gondii which is widely prevalent in humans and animals worldwide. The diagnosis of toxoplasmosis and distinguishing acute or chronic T. gondii infections have utmost importance for humans and animals. The TgSAG1, TgGRA7, and TgBAG1 proteins were used in the present study to develop the serological rSAG1-ELISA, rGRA7-ELISA and rBAG1-ELISA methods for the testing of T. gondii specific IgG and IgM antibodies and differentiating acute or chronic toxoplasmosis in 3733 animals, including Tibetan sheep, yaks, pigs, cows, cattle, horses, chickens, camels and donkeys from the Qinghai-Tibetan Plateau. The ELISA tests showed that the overall positivity of IgG antibody was 21.1% (786/3733), 15.3% (570/3733) and 18.2% (680/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively, and the positivity of IgM antibody was 11.8% (439/3733), 13.0% (486/3733) and 11.8% (442/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively. A total of 241 animals (6.5%) positive for all rSAG1-, rGRA7- and rBAG1-IgG were found in this study, and the 141 animals (3.8%) tested were anti-T. gondii IgM positive in all three ELISAs. Moreover, the 338, 284 and 377 animals were IgG positive in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1- ELISAs respectively, and the 346, 178 and 166 animals in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1-ELISAs were IgM positive respectively. The results confirmed that the application of SAG1, GRA7, and BAG1 recombinant antigens could successfully be used in the detection of specific IgG and IgM antibodies for distinguishing between acute or chronic T. gondii infections. It is inferred that the forms in which current animal species in the plateau area were infected with T. gondii, and the period of infection or the clinical manifestations of the current infections may be different. The present study provides substantial clinical evidence for the differential diagnosis of toxoplasmosis, and the classification of acute and chronic T. gondii infections.

Project description:BackgroundThe aim of the present study was to develop a simple, portable, and rapid assay for serodiagnosis of toxoplasmosis based on recombinant Toxoplasma gondii (T. gondii) SAG1 (rSAG1) and GRA7 (rGRA7) proteins.MethodsThe rSAG1 and rGRA7 proteins were expressed in Escherichia coli (E. coli) and purified in a single step by immobilized metal ion affinity chromatography. The immunoreactivity of the recombinant antigens was tested in an in-house IgG and IgM Dot enzyme-linked immunosorbent assay (Dot-ELISA) for potential use in serodiagnosis of T. gondii infection.ResultsResults from the comparison of in-house rSAG1-Dot-ELISA with ELISA for the detection of anti-Toxoplasma IgG and IgM include sensitivity of 83.7% and 81.2%, specificity of 90.2% and 89.3%, positive predictive values of 85.9% and 68.4%, and negative predictive values of 88.6% and 94.3%, respectively. Sensitivity of 66.2%, specificity of 81.2%, positive predictive values of 71.6%, and negative predictive values of 77.1% were concluded from in-house IgG rGRA7-Dot-ELISA. The sensitivity and specificity of IgM rGRA7-Dot-ELISA included 87.5% and 83.9%, respectively. Sensitivity and specificity of in-house Dot-ELISA for a combination of rSAG1 and rGRA7 included 87.5% and 91.1% for IgG and IgM, respectively. Sensitivity and specificity of a combination of rSAG1 and rGRA7 for the detection of IgM in suspected sera to acute toxoplasmosis were higher than those for the detection of IgG in sera with chronic infections (90.6% and 92% instead of 86.2% and 91.6%, respectively).ConclusionThe highlighted parameters of combined recombinant proteins were more significant than those of single recombinant proteins in in-house Dot-ELISA. These data suggest that the in-house Dot-ELISA based on rSAG1 and rGRA7 combination is a promising diagnostic tool with a similar sensitivity to the native antigens of T. gondii, which can be used for the serodiagnosis of toxoplasmosis in fields as well as less equipped laboratories.

Project description:BackgroundFew bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none is commercially available.ResultsWe report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1's N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98-100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen.ConclusionsOur recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies and ligands.

Dataset Information

Enhancing the detection of Toxoplasma gondii via an anti-SAG1 scFv-alkaline phosphatase immunoconjugate.

Publications

Enhancing the detection of <i>Toxoplasma gondii via</i> an anti-SAG1 scFv-alkaline phosphatase immunoconjugate.

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