Recombinant SAG1 antigen to detect Toxoplasma gondii specific immunoglobulin G in human sera by ELISA test

Although some serological tests for the detection of Toxoplasma gondii-specific immunoglobulin are commercially available, better diagnostic tools are needed. The aim of present study was to evaluate the usefulness of the recombinant Toxoplasma gondii SAG1 antigen for the recognition of toxoplasmosis by ELISA. This study was conducted in Cellular and Molecular Biology Research Centers, Shahid Beheshti University, M.C., Tehran, Iran in 2008-2009. Surface antigen 1 [SAG1], a tachyzoite stage-specific protein, was subcloned into an expression vector and was subsequently transformed into BL21 [DE3] pLyss competent bacterial cells. After inducing expression of the recombinant antigen, the protein product was purified using Ni-affinity chromatography. The immunoreactivity of recombinant SAG1 [rSAG1] was analyzed by SDS-PAGE and western blotting. The reactivity of the rec-SAG1 protein was evaluated using an ELISA. Sensitivity and specificity of the generated recombinant-ELISA [rec-ELISA] compared to a commercially available ELISA [com-ELISA] were 88.4% and 88%, respectively. Recombinant SAG1 produced in E. coli is a promising antigen that can be used in diagnostic assays for the detection of specific antibodies against T. gondii

[Evaluation of the second fraction of excreted/secreted antigens from toxoplasma gondii by ion exchange chromatography for serodiagnosis of toxoplasmosis in rat]

According to the previous studies, Toxoplasma gondii excreted/secreted antigens [E/SA] appear to be suitable marker for serodiagnosis of toxoplasmosis. Most of the previous studies used whole E/SA. The present study was carried out to evaluate the ELISA method using E/SA components from Toxoplasma gondii for the diagnosis of toxoplasmosis. Components obtained by incubation of tachiziotes at RPMI-1640 culture medium were purified by Ion exchange chromatogeraphy and fractions were analyzed by native-PAGE electrophoresis. Forty noninfected rats were injected as IP with 4x106 Toxoplasma tachyzoites and their serum samples were collected at 8, 15, 22 and 60 days after infection then were tested by dye-test. Based on these results the sera of 15 and 60 days were selected and tested by ELISA using E/SA. Second fraction of chromatography was selected as antigens. The cut-off point of ELISA with 99% confidence was found to be 0.41. Optical density of all sera samples of 15 and 3 of 60 days after infection and 2 negative sera were over the test cut-off. Therefore sensitivity and specificity of the method were determined to be 100% and 91% respectively. These results indicated that the second fraction of Ion exchange chromatography of Toxoplasma E/SA under these conditions may be useful tool for the serediagnosis and differentiation of acute and chronic phases of toxoplasmosis

Prevalence of helicobacter pylori infection in subjects with gastric cancer surgery

To determine the prevalence of helicobacter pylori [H. Pylori] infection in patients with gastrectomy for gastric cancer. Samples from 95 [69 males, 26 females] consecutive patients who had undergone surgery for gastric cancer were included in this study. Histologic examination of tumor obtained at the time of surgery yielded the diagnosis and then the specimens were stained with haematoxylin and eosin and Giemsa for the assessment of tumor and H.pylori infection. All patients did H.pylori immunologic and urease test. From 95 patients with gastric cancer, 83 patients [87.36%] were positive for H. pylori infection. Histologic type of tumors was intestinal in 61 patients and diffuses in 7 patients. The prevalence of H. pylori was 93.2% in intestinal type carcinoma and 28.5% in diffuse type [P- value=0.0002]. No significant difference was found in sex, smoking and staging between H.pylori positive gastric cancer and H.pylori negative cases. This study reconfirmed a high prevalence of H. pylori infection in patients suffering from gastric cancer and provided that evaluation for H. pylori infection might confer additional benefit in identifying the population that is at greater risk for this tumor

[Designing of ELISA kit for diagnosis of anti-toxoplasma specific IgG, using exerted/secreted antigens and its diagnosis potency]

Many studies report that Toxoplasma gondii excreted / secreted antigens [E/SA] appear to be a suitable marker for toxoplasmosis serodiagnosis. Most of these studies have used E/SA, obtained from supernatant of Toxoplasma cell culture, or by incubating tachyzoites in cell free media [RPMI-1640]. The present study for the detection of Toxoplasma IgG in human serum was evaluated, using the components of peritoneal fluid of infected mice [as another source of E/SA]. Peritoneal fluids of mice infected by interaperitoneal [IP] inoculation of Toxoplasma tachyzoites were collected after 3 days and centrifuged at 750 x g for 15 minutes. Then the supernatant was precipitated with ammonium sulphate solution [40% saturated] and used as components encompassing E/SA. Forty [40] none- infected [without anti- toxoplasma antibodies] and thirty- two [32] positive [with IgG to toxoplasma] human serum samples were selected [all sera were first tested by standard method for detection of IgG antibodies anti-T. gondii] then, the sera were tested by ELISA using E/SA. The cut-off point with 95% confidences was found to be 0.78. Moreover, sensitivity and specificity of the method were determined to be 84% and 92%, respectively. The present results indicate that peritoneal exudates from mice infected with T.gondii, may be used as a source of anti-genic material for the detection of Toxoplasma-specific IgG. Furthermore, it may be valuable for the development of new tools in the serodiagnosis of toxoplasmosis