ELISA-based Quantification of Anti-ASFV Antibodies

Anti-ASFV antibodies in the sera of infected animals were quantified using an in-house developed ELISA as previously described [9 (link)]. Briefly, ASFV was amplified in Vero cells [16 (link)], infected cells were then treated with Tween 80 (G-Biosciences, Saint Louis, MO, USA) and sodium deoxycholate (Sigma, Saint Louis, MO, USA) to a final concentration of 1% (v/v) and cell antigens were stored at ≤−70 °C. Maxisorb ELISA plates (Nunc, Saint Louis, MO, USA) were coated with 1 µg per well of either infected cell or uninfected cell antigen and blocked with phosphate buffered saline containing 10% skim milk (Merck, Kenilworth, NJ, USA) and 5% normal goat serum (Sigma). Swine sera were tested at multiple dilutions against both infected and uninfected cell antigen, with reactivity detected by an anti-swine IgG–horseradish peroxidase conjugate (KPL, Gaithersburg, MD, USA) and SureBlue Reserve peroxidase substrate (KPL). Plates were read at OD630 in an ELx808 plate reader (BioTek, Shoreline, WA, USA). Swine sera were considered positive for ASFV-specific antibodies if the OD630 ratio of the reaction against infected cell antigen to uninfected cell antigen was higher than 2.2.