To determine the antibody titer, an indirect ELISA test was performed as described by El Kojok et al. [36 (link)], with slight modifications. The ELISA plate was coated with 50 μL/well of recombinant antigen (from 1 µg/mL to 0.001 µg/mL) and/or virus sample (from 1:10 to 1:1000) diluted in 0.05 M carbonate buffer at pH 9.6 and incubated overnight at 4 °C. As a negative control, some wells were coated by coating buffer. The wells were washed three times with washing buffer (TBS 0.01 M pH 7.4 containing 0.05% Tween-20; TBST) and after the incubation with 200 μL/well of blocking buffer (TBS supplied with 5% w/v non-fat dried milk), at 37 °C for 2 h, the plate was rinsed three times. After this step, 50 μL/well of polyclonal antibodies, anti-ASFV (1 μg/mL) diluted in TBS, 1% non-fat dried milk, and 0.05% v/v TWEEN 20 buffer, were incubated at 37 °C for 2 h. The plate was rinsed (three times), 50 μL/well of goat anti-rabbit IgG-HRP antibody (0.5 μg/mL), was added, and the wells were incubated for 1 h at 37 °C. Finally, the enzyme substrate solution (TMB) was added (100 μL/well), and the wells were incubated at 37 °C, then the color development was quenched by adding stopping solution HCl (2.5 M 50 μL/well) after 10 min. A Tecan Infinity 200 Pro (Tecan, Männedorf, Switzerland) micro-plate reader was used to measure the absorbance at 450 nm.
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