Goat anti porcine igg h l af488

The presence of antibodies to individual proteins was determined using indirect immunofluorescence. Sera were used at a single dilution of 1:100 and incubated with Vero cells transfected with each of the individual genes fused to a V5 tag. Pre-immunization and pre-challenge sera were tested from pigs immunized with the pools of viral vectors expressing ASFV ORFs. Only pre-challenge sera from pigs immunized with the control antigens were analyzed for antibodies to ASFV proteins. Mouse anti-V5 tag (AbD Serotec, Oxford, UK. MCA1360) diluted 1:1000 was used as a positive control for V5 fusion gene expression. In indirect immunofluorescence, the anti-V5 antibody was incubated simultaneously with the animal sera on transfected cells. Secondary antibodies were goat anti-mouse IgG (H+L)-AF594 (Thermo Fisher, Hemel Hempstead, UK) and goat anti-porcine IgG (H+L)-AF488 (Southern Biotech, Birmingham, UK), all added at a dilution of 1:1000 to the cells. The cells were then observed under the fluorescence microscope and screened for simultaneous green and red fluorescence, resulting from recognition by specific antibodies in the pig serum of the expressed viral protein and the presence of the V5 tag respectively.