Fig. 3
Antibody titers produced in sera of immunized mice recognizing PRRSVchim protein. Groups were immunized with 25 μg of TEV VLPs (TEVVLPs25), 10 μg of PRRSVchim protein (PRRSV10), 10 μg of TEV VLPs plus 10 μg of PRRSVchim protein (TEVVLPs10/PRRSV10), 25 μg of TEV VLPs plus 10 μg of PRRSVchim protein (TEVVLPs25/PRRSV10) or 10 μg of PRRSVchim protein plus incomplete Freund’s adjuvant (PRRSV10/IFA). Immunization was carried out via subcutaneous with 100 μl of the different preparations on days 1 and 14. Blood samples were collected by tail vein bleeding before immunization and on day 28 after first immunization. Preimmune serum samples were checked for seronegativity to PRRSVchim protein. Serum samples collected from the second bleeding were tested for IgG, IgG1, IgG2a and IgG2b antibody response to PRRSVchim protein by ELISA. Individual samples belonging to the same group were pooled and diluted 1:20 followed by serial 1:5 dilution in blocking buffer. 96-well MaxiSorp Immuno Plates (Nunc, USA) were coated with 1 μg of PRRSVchim protein per well diluted in carbonate buffer, blocked and washed. Diluted serum samples were added to the appropriate well. After incubation and washing steps, the appropriate secondary antibody was added, HRP-donkey anti mouse IgG (Bio-Techne, USA) diluted 1:1,000 or HRP-goat anti mouse IgG subclass 1, 2a or 2b (JIR Laboratories, USA) diluted 1:5,000. After incubation and washing steps, 3-3′, 5-5′ tetrametylbenzidine (TMB) solution (Sigma-Aldrich, USA) was added to each well. Color development was monitored before saturation and stopped using sulfuric acid. Absorbance was taken at 450 nm in xMark spectrophotometer microplate reader (BioRad, USA). Results are expressed as antibody endpont titers greater than threefold the background value of preimmune sera