Goat anti mouse hrp conjugated serum

The ELISA was essentially performed as described previously [74 (link)], using lysed Vero cells previously infected with 5 PFU FPenvM766 as the plate-bound antigen. Briefly, 96-well maxisorp microtitre plates (Nunc, Naperville, IL, USA) were coated with Vero lysates in PBS^- (5 ×10⁴ cells/well) in 0.05 M carbonate–bicarbonate buffer, pH 9.6, and incubated overnight at 4°C. The sera from all of the animals, drawn at T0 and at different times p.i. (T1, T2), were then added at 1:20 dilution, and the binding was revealed using goat anti-mouse HRP-conjugated serum (1:2000 dilution; Dako) and tetramethylbenzidine substrate (Sigma). The absorbance for each well was measured at 450 nm with a microplate reader (550; Bio-Rad Laboratories, Hercules, CA, USA).

To determine whether the gp, env and CIITA proteins were expressed, CEFs and Vero cells were infected using the single and double recombinants (10 PFU/cell) and examined by WB, after loading the same amount of proteins, as described previously [69 (link), 70 (link)]. The experiments were repeated twice with similar results. Co-infection with single recombinants was also performed with Vero cells using 8 PFU/cell/each recombinant. The blotted nitrocellulose membranes were incubated overnight at 4°C using specific antibodies. For gp and env, anti-SIV monkey polyclonal antibodies were used (1:100-dilution; kind gift from J. Heeney, Department of Veterinary Medicine, University of Cambridge, UK), followed by rabbit anti-monkey horseradish-peroxidase (HRP)-conjugated serum (1:2,000 dilution; Sigma, St Louis, MO, USA). The anti-CIITA mouse monoclonal antibody (mAb) (1:500-dilution; 7-1H; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used, followed by goat anti-mouse HRP-conjugated serum (1:1,000 dilution; DakoCytomation, Carpinteria, CA, USA). After a 1-h incubation and 2-h washes, the proteins were revealed using the ECL system (EuroClone, Pero, Milan, Italy). Cells infected with FPwt were used as the negative control.