After the plates were blocked with 5% fetal bovine serum-PBS, 1 800 diluted (with blocking buffer) mouse sera were added to duplicate wells and incubated for 2 hours at room temperature.
Antibodies and blocking sera were diluted in 0.1% Triton X-100 in PBS.
After blocking, neosporosis-negative or -positive cattle sera were added, followed by anti-bovine antibody HRP, and detected by the addition of 3,3',5,5'-tetramethylbenzidine 3,3',5,5'-tetramethylbenzidine
Test or reference sera were added in 1 20 dilution in blocking buffer (PBS/0.05% Tween 20/5% non-fat milk/10% equine sera/0.1% E.coli 537 extract) and incubated for 30 minutes at 37°C.
After washing and blocking with 5% BSA in PBS for 2 hours, sera were added in serial dilutions, starting at 1/100 and 1/10, respectively, and were incubated for 1 hour at room temperature.
Following nonspecific protein binding blocking, a reference (CDC1992, NIBSC), a quality control, and unknown sera were added in duplicate and eight 2-fold dilutions made directly in the plate.
After blocking for 1 hour with 2% milk in PBS at room temperature, sera were added in serial dilutions in 2% milk/PBS and incubated overnight at 4°C.
After washing three times with PBST, sera were added at an initial dilution of 1 100 with a two-fold dilution series in blocking buffer and incubated for 2 h at 37 °C.
After the coated plate was blocked with 3% bovine serum albumin in PBS for 2 h at room temperature (RT), serially diluted sera were added to the plate and incubated at RT for 2 h.
