Western blot analysis of stray cat sera against Toxoplasma gondii and the diagnostic availability of monoclonal antibodies in sandwich-ELISA

A cDNA of 1.1 kb comprising the gene encoding the peroxiredoxin of Toxoplasma gondii (TgPrx) has been cloned. The open reading frame of 591 bp was translated into a protein of 196 amino acids with a molecular mass of 25 kDa. Conserved 2 cysteine domains of Phe-Val-Cys-Pro and Glu-Val-Cys-Pro indicated TgPrx belonged to 2-Cys Prx families. TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9% followed by Entamoeba histolytica with 39.5% by the amino acid sequence alignment. Polyclonal antibody against recombinant TgPrx detected 25 kDa band in T. gondii without binding to host cell proteins. TgPrx was located in the cytoplasm of T. gondii extracellularly or intracellularly by immunofluorescence assay. The expression of TgPrx was increased as early as 30 min after the treatment with artemisinin in the intracellular stage, while no changes in those of host Prx I and TgSOD. This result implies that TgPrx may function as an antioxidant protecting the cell from the attack of reactive oxygen intermediates. It is also suggested that TgPrx is a possible target of chemotherapy.

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“…Tachyzoites were purified by centrifugation over 40% Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) in PBS solution (Sohn and Nam, 1999).…”

Section: Parasitementioning

confidence: 99%

Molecular cloning and characterization of peroxiredoxin from Toxoplasma gondii

et al. 2001

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“…Several serological tests have been developed to detect specific antibodies to T. gondii in humans and animals. One of the immunologic tests using specific monoclonal antibodies (mAbs) was shown to be useful in detecting specific Toxoplasma antigens (Bonhomme, 1990;Sohn and Nam, 1999). The immunodeterminant proteins of Toxoplasma would be responsible for the immune response in host tissue; therefore, the purification and the characterization of these antigens should prove to be of a great value for diagnostic purposes.…”

Section: Introductionmentioning

confidence: 99%

Toxoplasma gondii: Ultrastructural localization of specific antigens and inhibition of intracellular multiplication by monoclonal antibodies

et al. 2001

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This experiment was focused on the characterization of anti-Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M621 were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs, M110, M556, R7A6 and M621, were 0.53, 0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgG1 isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that M110, M556, R7A6 and M621 reacted with the 33 kDa (p30), 31 kDa (p28), 43 kDa and 36 kDa protein. Immunogold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM), rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoites with four mAbs, M110, M556, R7A6 and M621 resulted in the decrease of the number of infected macrophages (p < 0.05) and the suppression of parasite multiplication at 18 h postinfection. Four monoclonal antibodies including M110 (SAG1) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy.

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“…As the seroprevalence of T. gondii in cats varied depending on their living type (stray or domestic), age, the diagnostic method used, and geographic area [13], the prevalence of T. gondii in stray cats has been reported to be variable from 5% to 45% in recent years in Korea [9][10][11].…”

mentioning

confidence: 99%