Rapid Detection of Toxoplasma gondii Antigen in Experimentally Infected Mice by Dot- ELISA (original) (raw)
Related papers
Detection of Toxoplasma gondii antigens by a dot-immunobinding technique
Journal of clinical microbiology, 1985
A sensitive assay for the detection of antigens of Toxoplasma gondii by spotting samples directly onto nitrocellulose paper was developed. The sensitivity ranged from 10 to 40 pg of antigen diluted in phosphate-buffered saline and 40 to 130 pg of antigen diluted in normal mouse serum, normal human serum, or human cerebrospinal fluid. T. gondii antigen in serum samples taken from mice infected with T. gondii was detectable by day 2 of infection. Antigen was also detectable in cerebrospinal fluid samples taken from four of six infants congenitally infected with T. gondii and in serum samples from two of these infants.
Clinical and diagnostic laboratory immunology, 1996
Different techniques for identifying Toxoplasma gondii were compared. PCR was used to amplify part of the major surface antigen P30 gene of T. gondii. Amplified-DNA detection with the DNA enzyme immunoassay (PCR-DEIA) was more sensitive than ethidium bromide staining after agarose gel electrophoresis and as sensitive as nested PCR. PCR-DEIA, using common enzyme-linked immunosorbent assay (ELISA) methods, avoids agarose gel electrophoresis for the identification of amplified products. T. gondii can also be detected with equal sensitivity in infected fibroblasts, but only after at least 8 days of cell culture. PCR-DEIA is thus recommended because of its sensitivity and convenience for detecting early parasitemia in the surveillance of toxoplasmosis among pregnant women and immunocompromised hosts. The courses of infection in mice infected with two strains of T. gondii were compared. Tachyzoites of the virulent strain T. gondii RH, killing the host in 4 days, were identified in urine specimens and blood samples of mice 24 to 94 h after inoculation but not in brains, but no antibodies were detected. After intraperitoneal inoculation with cysts of the low-level virulence Beverley strain of T. gondii, parasites were identified in blood samples 4 days later and up to 17 days (but not in urine specimens) and in the brain from day 6 through day 525. By ELISA, high antibody titers were found from day 11 to day 525, with parasitemia preceding the appearance of antibodies. The usefulness of PCR-DEIA tests in conjunction with the search for circulating antibodies for the early diagnosis of toxoplasmosis in humans is discussed. In congenital infection, Toxoplasma gondii may cause fetal death or severe neurological sequelae, such as hydrocephalus, microcephalus, or blindness (25). Fetal contamination is estimated to occur in 0.1 to 0.5% of infected pregnant women in the United Kingdom (1), 0.2 to 0.3% in southern Finland (19), 1% in France (24), 0.2 to 0.8% in Canada (4), and 0.2 to 0.6% in the United States (11). Prevention of congenital toxoplasmosis in pregnant women has been based mainly on serological tests for anti-Toxoplasma antibodies. In acquired diseases, specific antibodies belong to the immunoglobulin M (IgM), IgA, and IgG classes (6, 8, 28, 30). However, infection should be diagnosed at the early acute stage, when treatment is more effective. In this study, PCR, cell culture, and enzyme-linked immunosorbent assay (ELISA) were used to detect T. gondii in urine specimens, blood samples, and brains of mice experimentally infected by virulent and weakly virulent strains of T. gondii and were compared. Our data suggest that PCR-DNA enzyme immunoassay (PCR-DEIA) is a highly sensitive method for detecting this parasite in biological samples. Since PCR-DEIA is as sensitive as cell culture but much more convenient and rapid, it should be used for the early diagnosis of human congenital toxoplasmosis. MATERIALS AND METHODS Mice and T. gondii infection. NMRI female mice (6 to 8 weeks old) were used in these experiments. Mice were obtained from the animal facility of Catholic University of Louvain, Brussels, Belgium. Sera were tested by ELISA (5) to confirm the absence of T. gondii antibodies before the experiments. Cysts of the low-level virulence Beverley strain of T. gondii were obtained from chronically infected NMRI female mice. The source of the Beverley strain was isolated by J. K. A.
2014
2 Abstract: Toxoplasmosis can be diagnosed indirectly with serological methods with detection of specific Toxoplasma IgG, IgM and IgA antibodies, yet those techniques are not reliable enough to detect specific anti-T. gondii antibodies during the active phase of the parasitic infection. In the current study, we have purified a specific T. gondii antigen (s) as to detect its efficacy in diagnosis of the disease at the hardly detectable phase. ELISA and immunoblot assays in parallel with a developed home-made dot ELISA were used to achieve our goal. A total of 153 human sera were collected from patients and assigned into three groups: A first group of 88 sera pooled from T. gondii-infected patients,a second group of 35 samples collected from patients infected with other parasites and, ultimately, a third group of 30 sera pooled from healthy individuals (control). Our results showed a comparable significance in the sensitivity, specificity and efficacy obtained by using the commercial ...
A New Alternative In Vitro Method for Quantification of Toxoplasma gondii Infectivity
Journal of Parasitology, 2012
An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25cm 2 flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1:10 6 to 1:10; we defined 10 6 tachyzoites/ml 21 as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 10 6 tachyzoites/ml 21. Finally, we validated the method using a known mixture of dead and live parasites. This method was found to estimate with accuracy the initial ratio of infection of the unknown parasite suspension. This easy-to-use method is reproducible and can be applied to any T. gondii tachyzoite RH strain, genetically modified or not. This method is also suitable for testing promising candidates for an effective live vaccine.
Journal of Parasitic Diseases, 2019
Toxoplasma gondii (T. gondii) is a worldwide distribution infects a wide variety of mammals, including humans. The present study aimed to detect the efficacy of soluble and whole T. gondii antigens propagated in specific pathogen-free of embryonated chicken egg (SPF-ECE) used to improve the potency of serological assays for diagnosis of toxoplasmosis in equids and human. Total of 220 serum samples from 170 equids (90 donkeys and 55 horses and 25 mules) and 50 humans were collected from different governorates in Egypt during the period from October 2017 to March 2018. Crude T. gondii tachyzoites antigens from low or high passages propagated in mice or SPF-ECE was used for modifying some serological tests. The experiment showed that the mortality rate of T. gondii for 10 3 and 10 4 low passages were 6/8 (75%) and 7/8 (88%) dead embryos but, lower mortality rate in high passage T. gondii were 4/8 (50%) and 5/8 (63%) dead embryos, respectively. No mortality or inflammatory signs were observed in control of negative groups. In equids sera were examined by S-ELISA using soluble T. gondii antigen propagated in SPF-ECE showed the highest positive results 26 (28.8%), followed by LAT 37 (22%) and MAGPT 36 (21.17%). While, W-ELISA and IFAT used whole T. gondii antigen prepared in SPF-ECE were 35 (20.58%) and 28 (19.41%) showed highly positive results than the same test used the whole antigen prepared in mice. The highest seroprevalence of T. gondii in human and donkeys were 19/50 (38%). and 26/90 (28.88%), more than mules were 6/25 (24%) and horses were 9/55 (16.3%) examined by S-ELISA respectively. SPF-ECE is considered an appropriate experimental model for isolation and propagation of T. gondii tachyzoites, and their soluble antigens used in serological tests (S-ELISA, LAT, and MAGPT) have sensitivity and specificity more than the whole antigen and provided reliable diagnostic tools for detection of toxoplasmosis in human and equids.
Detection of antibodies to the 97 kDa component of Toxoplasma gondii in samples of human serum
Memorias Do Instituto Oswaldo Cruz, 2002
This study was carried out to investigate the immune response against 97 kDa (p97) molecular marker of Toxoplasma gondii that has been characterized as a cytosolic protein and a component of the excreted-secreted antigens from this parasite. A total of 60 serum samples from patients were analyzed by enzime-linked immunosorbent assay and Western blot for toxoplasmosis. These samples were organized in three groups, based on clinical symptoms and results of serological tests. Group I: 20 samples reactive to IgG and IgM (acute phase); group II: 20 nonreactive samples (control group); and group III: 20 samples reactive only to IgG (chronic phase). Western blot was performed with total antigenic extracts or with excreted and secreted antigen from T. gondii to identify the fraction correspondent to p97. It was observed that this cytosolic component from T. gondii stimulates the immunologic system to produce both IgM and IgG antibodies in the beginning of the acute infection and IgG throughout the chronic stage of the asymptomatic toxoplasmosis.
The Tohoku Journal of Experimental Medicine, 1985
A one point dilution enzyme-linked Immunosorbent assay (ELISA) procedure suitable for determining immunoglobulin G (IgG) antibody levels to Toxoplasma gondii (T. gondii) in community seroepidemiological surveys is described. A twofold serial dilution ELISA procedure was first used to determine the IgG titers in 56 and 83 sera earlier screened by the Sabin-Feldman dye test (DT) and the indirect hemagglutination test (IRA), respectively. The regression rate of the results by the DT and ELISA was 0.92. Comparison of the results by the IHA and the twofold serial dilution ELISA gave regression coefficient of 0.92. Using the absorbance values for the test sera at dilutions of 1: 20, standard curves made by plotting the optical density versus the corresponding dilution factor of a control sera were used to estimate the antibody levels. The regression coefficient of the results by the twofold serial dilution method and those by the curves for sera with titers of up to 1: 320 was 0.97. The curves could not, however, estimate accurately the antibody level in sera with titers above 1: 320. The one point dilution ELISA described is a useful epidemiological tool for the screening of IgG antibody to Toxoplasma gondii in the community. However, larger series are required to confirm our observations.-Toxoplasma gondii ; ELISA ; seroepidemiological survey A number of serological tests have been described for the detection of antibody to Toxoplasma gondii. In choosing a diagnostic test, however, the specificity, sensitivity, safety, cost and time required to get the results are some of the major determining factors. In this regard, the enzyme linked immunosorbent assay (ELISA), has been noted to offer a combination of the best qualities (Wisdom 1976). Although originally described as a single tube, photometrically
Experimental Parasitology, 2006
Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7 · 10 7 viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days À6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10 = 2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as ''gold standard'' and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISA · IFAT (j = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 ± 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs.
An improved assay for the detection of Toxoplasma gondii antibodies in human serum by flow cytometry
Cytometry, 1993
The diagnosis of toxoplasmosis relies primarily on the demonstration of specific antibodies to Toxoplasma gondii. We describe a flow cytometry method for the determination of antibodies to whole fixed tachyzoites by indirect immunofluorescence. Fixed tachyzoites in suspension have characteristic light scattering properties. The amount of IgM, IgG, and IgA antibodies from patients' sera bound to the tachyzoites can be estimated from the mean fluorescence intensities observed using class-specific conjugates for different Ig heavy chains. Appropriate serum dilutions for the estimation of specific IgG titres and for the discrimination between sera positive and negative for IgA and IgM antibodies were established using 40 random sera from pregnant women. The method proved to be quantitative and highly sensitive as compared with currently used assays. Coefficients of variation between series ranged from 6.4% to 12.2% and could be controlled by the inclusion of positive and negative st...