Strips were cut and incubated with serum from the rabbits (diluted 1 : 100 in 1% BSA/PBS) for 2 hr

Strips were cut and incubated with serum from the rabbits (diluted 1 : 100 in 1% BSA/PBS) for 2 hr. antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis. Keywords: antigen in the serum or other body fluids of patients would help to diagnose correctly and prevent late complications. For detecting antigen, several laboratory procedures are available. Direct detection procedures, such as microscopic examination, immune histology, or cell culture are reliable, but they are either insensitive or time-consuming [1,2]. PCR is highly sensitive and specific, although heme, heparin, and other poorly characterized substances have been reported to decrease the efficiency of PCR [3]. ELISA is considered to Sulcotrione be a highly sensitive, practical method for detecting the parasite antigen [2]. Many reports have discussed titrating serum antibodies in hosts after infection, however, little information is available on the correlations among parasitemia, circulating antigens, and antibody titers in subcutaneously. Then, blood samples were drawn from an ear vein of each rabbit every other day for 20 days. To check parasitemia in the rabbits, 0.5 ml of heparinized blood from each rabbit Sulcotrione was injected intraperitoneally into 4 mice, and their survival was monitored for 20 days after infection. The ELISA for detecting circulating antigens was performed in microtitration trays [4,5]. To obtain mouse anti-antisera, mice were infected with 20 brain cysts of avirulent Me49 strain of orally. The mice were then sacrificed at 6 months after infection, and the sera were precipitated with saturated ammonium sulfate solution, resuspended in 0.01 M phosphate buffered saline. Mouse anti-antisera were diluted with 0.1 M carbonate-bicarbonate buffer (pH 9.6, 10 g/ml). Then, 100 l were pipetted into 96-well microtiter plates (Nunc, Roskilde, Denmark) and incubated at 4 overnight. The plates were washed with PBS containing 0.05% Tween 20 (PBS/Tween 20), to which 0.1 ml of rabbit serum diluted 1 : 50 with PBS/Tween 20 containing 0.1% bovine serum albumin was added. lysate antigen (TLA) was prepared as a control. The plates were incubated at room temperature (RT) for 2 hr, and then 0.1 ml sample serum from the infected rabbit was added. After Sulcotrione washing, 150 l of horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Sigma Chemical Co., St. Louis, Missouri, USA) diluted 1 : 3,000 were added to each well, and then the plates were incubated for 2 hr at RT. Subsequently, the plates were washed with PBS/Tween 20, and 150 l of < 0.05. For immunoblotting, TLA was heated with sample buffer at 100 for 4 min, separated on 12% acrylamide separating gels under reducing conditions, and then transferred electrophoretically to nitrocellulose sheets (Schleicher & Schuell BioScience Inc., Dassel Germany) at a constant voltage of 50 V for 1 hr at 4. The nitrocellulose sheets were incubated for 2 hr with 5% nonfat powdered milk in PBS. Strips were cut and incubated with serum from the rabbits (diluted 1 : 100 in 1% BSA/PBS) for 2 hr. After 3 washes with PBS, the strips were incubated for 2 hr in HRP-conjugated goat anti-rabbit immunoglobulin (Sigma) diluted 1 : 5,000 in 1% BSA/PBS. After washing, the strips were incubated with 4-chloro-1-naphthol solution for 2 hr at RT. The reaction was stopped by rinsing with PBS. Two rabbit died on 8 to 10 days after infection, while the other 3 rabbits survived until the end of the experiment. For the determination of parasitemia, 4 mice of each group were inoculated intraperitoneally with 0.5 ml of infected rabbit blood. As shown in Fig. 1, the rabbits developed parasitemia beginning on day 2 post-infection (PI), and this peaked between Mouse monoclonal to CD4.CD4 is a co-receptor involved in immune response (co-receptor activity in binding to MHC class II molecules) and HIV infection (CD4 is primary receptor for HIV-1 surface glycoprotein gp120). CD4 regulates T-cell activation, T/B-cell adhesion, T-cell diferentiation, T-cell selection and signal transduction days 4 and 6 PI Sulcotrione (90 13% to 95 11%). Mice did not die after day 12 PI and it means that tachyzoites were not contained any more in the rabbit blood. Open in a separate window Fig. 1 Mouse inoculation test for detecting parasitemia in rabbits infected with the RH strain of.