Anti human igg conjugated with hrp horse radish peroxidase

Tachyzoites of T. gondii, RH strain (from School of Public Health, Tehran University of Medical Sciences) were harvested from mice peritoneum after 3 days of inoculation. Tachyzoites were washed, sonicated, centrifuged at 14000g for 1 hour; the supernatant was collected and the protein quantity was determined by method of Bradford (17 ).
Microtiter 96 well plates were coated with 100 μl containing 5 μg/ml of protein and kept at -20°C until use. For IgG ELISA, plates were washed with PBST (phosphate buffer saline, 5% tween), then blocked with blocking buffer (skimmed milk 2.5% in PBST). After incubation and washing, 100μl of diluted sera (1:200) in PBST were added to each well. After incubation and washing, 100μl of anti- human IgG conjugated with HRP (horse radish peroxidase) (DAKO, Denmark) in dilution of 1:1000 with PBST was added to each well and afterward performed incubation and washing. Then, chromogenic substrate OPD (ortho-phenylen-diamidine) (Merck, Germany) was added to each well. Enzymatic activity was obvious after 15 minutes. Reaction was terminated by adding of 20% sulfuric acid. Optical density was recorded at 492nm with an automated ELISA reader (BIOTEC, LX800, USA).