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Characterization Of Recombinant Surface Antigen-1 (Rsag-1) Of Toxoplasma Gondii Through Western Blotting Using Mouse Anti-Serum

By: Mati Ullah Khan (2008-VA-107) | Dr. Haroon Akbar.
Contributor(s): Prof. Dr. Khalid Saeed | Dr. Uzma Farid Durrani.
Material type: materialTypeLabelBookPublisher: 2015Description: 40p.Subject(s): Department of ParasitologyDDC classification: 2397-T Dissertation note: Toxoplasmosis is caused by Toxoplasma gondii, which is an obligate intracellular apicomplexan protozoan parasite. The infection is highly prevalent worldwide, and is important both from veterinary and human health concern. In the past recombinant surface antigen-1(rSAG-1) has been shown to be a good candidate for the development of immuno-diagnostic kits as well as Vaccine. Through immuno-blotting, Surface antigen-1 has been identified by sera collected from Toxoplasma gondii infected cats, dogs and humans. Looking at the importance of this health threatening issue, the current study was designed to characterize rSAG-1 through western blotting for development of local diagnostic kit through western blotting using mouse anti-serum. The rSAG-1 was previously expressed under the project of Grand Challenges Canada at Molecular Parasitology laboratory, Department of Parasitology, University of Veterinary and Animal Sciences, Lahore. Recombinant SAG-1 was quantified by using commercial kit based on BCA assay. The 15 μg of rSAG-1 was inoculated subcutaneously (S/C) 3 times with each 2 weeks interval in mice to raise hyper-immune serum. Blood was collected from mice two weeks after the each injection through lateral Retro-Orbital Bleeding (Sharma et al. 2014). Serum was collected by centrifugation. The rSAG-1 was electrophoresed on 12% polyacrylamide gel through SDS-PAGE technique and the protein was transferred to nitrocellulose membrane for western blotting. Anti-serum raised against rSAG-1 was cross-reacted with the rSAG-1 already immobilized on the nitrocellulose membrane. Anti-mice immunoglobulin G conjugated with Alkaline Phosphatase (AP) was used as secondary antibodies for the development of immuno-blot. Immuno-blot revealed a band of 35 KDa.
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Toxoplasmosis is caused by Toxoplasma gondii, which is an obligate intracellular apicomplexan protozoan parasite. The infection is highly prevalent worldwide, and is important both from veterinary and human health concern. In the past recombinant surface antigen-1(rSAG-1) has been shown to be a good candidate for the development of immuno-diagnostic kits as well as Vaccine. Through immuno-blotting, Surface antigen-1 has been identified by sera collected from Toxoplasma gondii infected cats, dogs and humans. Looking at the importance of this health threatening issue, the current study was designed to characterize rSAG-1 through western blotting for development of local diagnostic kit through western blotting using mouse anti-serum. The rSAG-1 was previously expressed under the project of Grand Challenges Canada at Molecular Parasitology laboratory, Department of Parasitology, University of Veterinary and Animal Sciences, Lahore. Recombinant SAG-1 was quantified by using commercial kit based on BCA assay. The 15 μg of rSAG-1 was inoculated subcutaneously (S/C) 3 times with each 2 weeks interval in mice to raise hyper-immune serum. Blood was collected from mice two weeks after the each injection through lateral Retro-Orbital Bleeding (Sharma et al. 2014). Serum was collected by centrifugation. The rSAG-1 was electrophoresed on 12% polyacrylamide gel through SDS-PAGE technique and the protein was transferred to nitrocellulose membrane for western blotting. Anti-serum raised against rSAG-1 was cross-reacted with the rSAG-1 already immobilized on the nitrocellulose membrane. Anti-mice immunoglobulin G conjugated with Alkaline Phosphatase (AP) was used as secondary antibodies for the development of immuno-blot. Immuno-blot revealed a band of 35 KDa.

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