Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay

Figure 1. Classical swine fever virus (CSFV) amplified luminescent proximity homogenous assay (AlphaLISA®) platform for detection of CSFV Erns IgG antibody. In the presence of Erns IgG in a tested sample (serum, oral fluid), the Erns IgG binds Erns protein that conjugated to acceptor beads, and the biotinylated anti-swine IgG that conjugated to streptavidin donor beads. Binding of the antibody brings the donor and acceptor beads into proximity. Excitation of the donor beads at 680 nm promotes the release of singlet oxygen molecules that trigger a cascade of chemical events in the acceptor beads, resulting in a light emission at 615 nm.

Figure 2. Optimization of biotinylated anti-swine IgG antibody concentrations for serum and oral fluid AlphaLISA®. The optimum combination of a mix containing 25 µg/mL of Erns protein-conjugated acceptor beads and 80 µg/mL of streptavidin donor beads were tested with various biotinylated anti-swine IgG concentrations: (a) negative and positive sera were evaluated at 10, 20, 40, 80, and 100 nM; (b) negative and positive oral fluids were tested at 20, 40, 80, 100, and 160 nM. The AlphaLISA® signal represents the mean values (n = 4) with error bars denoting the standard deviation.