Fitc conjugated goat anti mouse igg h l

The quantification of PCV2 titer was determined using the method described by Zhu et al. [26 (link)] with modifications. Total virus yield (supernatants from cell lysates) were determined by inoculating tenfold serial dilutions into confluent PK-15 cells in 96-well culture plates (Corning™ Cellgro™). After 72 h of incubation, supernatant was removed, cells were fixed with 80% cold acetone and the viral antigen was detected using immunofluorescence (IFA) with mouse anti-Cap PCV2-specific monoclonal antibody (isotype IgG2a, Jeno Biotech Inc.) and FITC-conjugated goat anti-mouse IgG (H + L) (Kirkegaard & Perry Laboratories Inc.). The 50% tissue culture infective dose (TCID₅₀) was calculated according to the Reed-Muench method and expressed as TCID₅₀/mL.

For immunofluorescence microscopy, 10 mg of freeze-dried chitosan microparticles were blocked overnight in 200 μl of PBS/5% skim milk at 4°C and then incubated overnight at 4°C with a mouse anti-Cap PCV2-specific monoclonal antibody (isotype IgG2a, Jeno Biotech Inc.) diluted 1:100 in PBS/0.1% Tween-20 (PBST). After washing with PBST, the microparticles were incubated with FITC-conjugated goat anti-mouse IgG (H + L) (Kirkegaard & Perry Laboratories Inc.) for 1 h. After further washing, the microparticles were visualized under a Nikon Eclipse E400 fluorescence microscope interfaced to a PC running capture software (Nis-Element Br, Nikon).
Microparticles loaded with extracts of S. cerevisiae transfected with an empty plasmid (S. cerevisiae/pYES2) were subjected to the same treatment and used as a negative fluorescence control.