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.