96 microwell plates

Antibody responses to p30 and p54 were measured by indirect ELISA. Briefly, 96-microwell plates (Costar, Cambridge, MA, USA) were pre-coated with recombinant p30 (0.125 µg/mL) or modified p54 (0.5 ug/mL) at 100 µL/well in coating buffer (0.1 M carbonate buffer, pH 9.0). After incubation overnight at 4 °C, each plate was blocked with 5% skim milk (200 μL/well) for 2 h at 37 °C and washed five times with PBST. Then, serum samples (1:100 dilutions) from immunized mice were added at 100 μL/well and incubated for 1 h at 37 °C. Positive, negative and blank controls were also established. Subsequent steps were performed in accordance with a conventional protocol. The OD at 450 nm (OD₄₅₀) of each well was detected using a microplate reader (Thermo Fisher Scientific). IgG1 and IgG2a were measured in the same way, except that serum samples were diluted in a twofold series and the secondary antibodies were HRP-labeled goat anti-mouse IgG1 or IgG2a (Abcam) instead of HRP-labeled goat anti-mouse IgG. IgG levels to p30 and p54 were expressed as OD₄₅₀ values. To calculate titers of IgG1 and IgG2a, a cutoff value was defined as the mean specific OD₄₅₀ value plus three standard deviations from mouse sera at 0 dpv, with a dilution of 1:100. The p30- and p54-specific IgG1 and IgG2a titers corresponded to the reciprocal values of the highest dilutions that showed positive.

Antigen-specific IgG responses in the sera from vaccinated pigs were measured by indirect ELISA. Briefly, 96-microwell plates (Costar, Cambridge, MA, USA) were pre-coated with recombinant p30 (0.125 µg/mL), modified p54 (0.5 µg/mL), p72 (1 µg/mL), △pE248R (1 µg/mL), △CD2v (2 µg/mL) and △pEP153R (2 µg/mL) overnight at 4 °C and blocked with 5% skim milk for 2 h at 37 °C. Serum samples (1:100 dilutions) from immunized pigs were added to each well and incubated for 1 h at 37 °C. In parallel, blank and negative controls were set up. After five washes with PBST, the plates were incubated with anti-pig IgG-HRP (1:10,000 dilutions) for 1 h at 37 °C. After a final wash series, color were developed with 3,3’,5,5’-tetramethylbenzidine substrate for 15 min at 37 °C and stopped by 2 M H₂SO₄. The OD at 450 nm (OD₄₅₀) of each well was determined using a microplate reader (Thermo Fisher Scientific).