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.