2.2 Virus-like particles (VLPs)
Recombinant VLPs were obtained as previously reported (Mignaqui et al.,
2013). Briefly, suspension-growing 293-6E cells were grown in serum-free
F17 medium (Gibco) at 37°C with 5% CO2 and agitation at
120 rpm. Cells were transiently transfected with pTT5-P12A3C plasmid
encoding for FMDV Serotype A/Arg/2001 VLPs using polyethylenimine
(LPEI-MAX) (Polysciences, Warrington, PA, USA). Cells were harvested 48
h post transfection, centrifuged at 4000 g and the pellet was
resuspended in Tris-HCl, pH 8, and subjected to three freeze-thaw cycles
at -80ºC / 25ºC. Finally, the lysate was clarified by centrifugation and
VLPs were quantified by ELISA, as previously reported (Mignaqui et al.,
2013). Briefly, a polyclonal anti-FMDV serum raised in rabbit was used
for coating microtiter plates (Maxisorp). After washing steps, plates
were blocked for 30 min at 37°C with 5% normal equine serum in
PBS/0.1% Tween-20. VLPs samples were added to the wells and incubated
at 37°C for 1 h. A standard curve was generated using serial dilutions
of a quantified aliquot of inactivated FMDV. Plates were then incubated
for 1 h with a polyclonal anti-FMDV serum raised in guinea pig, followed
by horseradish peroxidase-conjugated goat anti-guinea pig IgG (KPL).
Then, tetramethylbenzidine was used as substrate and the absorbance at
450 nm was recorded in a microplate reader (Thermo Scientifics
MultiskanFC).