2.8 Measurement of total FMDV-specific Abs by liquid phase ELISA
An lpELISA test was used according to Hamblin et al (1986), with modifications (Hamblin et al., 1986; OIE - World Organisation for Animal Health, 2012)\sout. Briefly, Greiner Microlon® plates were coated overnight at 4 oC with rabbit anti-FMDV serum diluted to a previously established optimal concentration in carbonate-bicarbonate buffer, pH 9.6. After washing with 0.05% Tween-20/phosphate buffered saline (PBST), plates were blocked with PBST/1% ovalbumin (blocking buffer) for 30 min at 37 °C. Mice or bovine sera were serially diluted (1:10) in blocking buffer in separate tubes and a fixed amount of inactivated FMDV was added. After 1 h incubation at 37°C with shaking, the virus-antibody mixtures were transferred to the blocked plates, and incubated for 1 h at 37°C. An optimal dilution of guinea pig anti-FMDV serum in PBS/2% normal bovine serum/2% normal rabbit serum was added for detection, followed by 1 h incubation at 37°C. Plates were washed and peroxidase-conjugated anti-guinea pig IgG (Jackson ImmunoResearch®) serum diluted in the same buffer was added, followed by 1 h incubation at 37°C. OPD/H2O2 was used as peroxidase substrate as above and A492 was measured in a microplate reader. Strong positive, weak positive and negative bovine reference sera were included in each test for validation. Antibody titers were expressed as the negative logarithm of the highest dilution of serum that causes an inhibition of color development higher than 50% in the average values of the control samples.