Seasonal recurrence of cowpox virus outbreaks in captive cheetahs (Acinonyx jubatus)

Stagegaard, Julia; Kurth, Andreas; Stern, Daniel; Dabrowski, Piotr Wojciech; Pocknell, Ann; Nitsche, Andreas; Schrick, Livia

For ELISA, PolySorp microwell plates (Nunc) were coated with 100 μL of lysate of infected or non-infected HEp-2 cells at 4 μg/mL in 0.1 M carbonate buffer (pH 9.6) over night at 4˚C. Between each step, plates were washed with 4 × 300 μL of washing buffer (100 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20). Plates were blocked at room temperature for 1 hour with 200 μL per well of 3% bovine serum albumin (BSA, Carl Roth) in washing buffer. Subsequently, 100 μL per well of non-inactivated sera were incubated at a 1:100 dilution in washing buffer supplemented with 0.25% BSA for 1 hour before detection with either goat anti-cat IgG (H+L) HRP, goat anti-dog IgG (H+L) HRP, or goat anti-mouse IgG (H+L) (Dianova, used at a 1:5000 dilution). Finally, signals were developed for 15 minutes by using 100 μL of Seramun-Slow TMB substrate (Diavita) per well before the reaction was stopped by addition of 100 μL of 0.25 M H 2 SO 4 . The resulting absorption was read at 450 nm referenced to 620 nm at an ELISA reader (Tecan). Each serum was tested in two replicate measurements on both infected and non-infected HEp-2 cell lysate. Binding signals against non-infected HEp-2 cell lysate were subtracted from signals against vaccinia virus-infected cells, and sera with a differential binding signal above 0.05 were considered positive.