2.7 Measurement of total IgG and isotypes against FMDV by sandwich ELISA
Total Abs against FMDV were assessed by ELISA as described previously (Batista et al., 2010; V Quattrocchi et al., 2011; Zamorano et al., 2010). Briefly, Greiner Microlon® plates were coated ON at 4°C with anti-FMDV rabbit serum in carbonate–bicarbonate buffer, pH 9.6. After three washing steps, plates were blocked for 30 min at 37oC with polyvinylpyrrolidone blocking solution in the case of mouse sera (0.5 M NaCl/ 0.01 M phosphate buffer/ 0.05% Tween-20/ 1 mM EDTA/ 1% polyvinylpyrrolidone 30–40 K, pH 7.2) or with PBS / 10% fetal calf serum (FCS) in the case of bovine sera. An optimal dilution of inactivated FMDV in blocking solution was added, followed by incubation at 37◦C for 30 min. Then, serially diluted mouse sera (1:4) or bovine sera (1:5) in blocking solution were added. After 1 h 20 min incubation at room temperature, plates were washed and an optimal dilution of horse radish peroxidase (HRP)-conjugated anti-mouse IgG (H+L) (KPL®), anti-mouse isotypes (Southern Biotech®), anti-bovine IgG (KPL®) or anti-bovine IgG1 or IgG2 (KPL®) were added. Plates were incubated for 1 h at room temperature and then washed. Ortho-phenylene-diamine (1,2-benzenediamine) dihydrochloride (SIGMA®) (OPD)/H2O2 was used as peroxidase substrate. Reactions were stopped using 1.25 M H2SO4 and A492 was measured in a microplate reader. Positive and negative control sera were included in every plate. The cut-off was established as the mean of the values of negative sera (n= 10) plus two standard deviations.