Tmb microwell peroxidase substrate system

Serum was collected at 0, 7, 14, 21, 28, 35, 42 and 49 days post-primary immunisation from the vaccinated pigs, and an indirect ELISA was used to detect SPV-specific antibodies. We used purified SPV in 100 μL of PBS (pH 7.2) at an empirically optimised dilution to coat 96-well plates. The virus was added to the plates and then incubated overnight at 4 °C. The following morning, the plates were washed three times with PBST and then blocked with 5% skim milk (in PBST) at 37 °C for 2 h. Serum samples were serially diluted and incubated at 37 °C for 1 h. The samples were set up at the same time and divided into the following five groups: (1) Δ003 positive serum, (2) Δ010 serum, (3) ΔTK serum, (4) wtSPV positive serum, and (5) blank control. After incubation, the plates were washed three times with PBST, and then horseradish peroxidase (HRP)-conjugated goat anti-SPA IgG (1:10 000 diluted in PBST, Signalway Antibody) was added to each well. The plates were incubated at room temperature in the dark for 30 min and then washed three times with PBST. The TMB microwell peroxidase substrate system (TIANGEN) was used to develop the reaction. Samples were developed for 20 min, and the reaction was stopped with 2.0 M sulfuric acid. All assays were performed in duplicate. A micro plate reader (Bio-Rad) was used to measure the reaction products at an absorbance of 450 nm [34 (link)].