Immunofluorescence assay
The in-house IFA uses OPXV-infected cells as target antigens and can detect IgM or IgG antibodies but does not distinguish neutralising antibodies. For the preparation of about 30 slides, 3×106 suspended HEp2 cells (ECACC) per mL in 15 mL of medium were pelleted for 5 min at 216×g. The pellets were resuspended in 1 mL of virus-containing medium and infected with VACV Lister-Elstree at MOI of 0.5 or CPXV HumGri07/1 at an MOI of 0.9 for 1 h at 37 °C, 5 % CO2. After mixing with 10 mL of medium, the cells were pelleted for 5 min at 216×g, resuspended in 3 mL of medium, and 30 µL each were applied to 12-well cavity slides (PTFE(Teflon)-coated, e.g., VWR #631-9423). A negative control with uninfected cells was included on each slide. The slides were incubated for 24 h at 37 °C, 5 % CO2, the supernatant was removed and the slides were air-dried. Subsequently, the cells were fixed in acetone for 60–90 min at room temperature, air-dried and stored at -20 °C until usage. Quality control of the slides was performed with known OPXV-positive controls for IgM and IgG. As standard procedure, all serum samples were inactivated for 30 min at 56 °C. For IgM detection, the serum was additionally pre-treated with Mastsorb Absorbens (Mast Diagnostika #651003) for 5 min at room temperature following supplier recommendations. Briefly, 75 µL of Mastsorb were mixed with 60 µL of PBS and 15 µL of inactivated serum were added. After 30 min of incubation at room temperature, the treated serum was centrifuged for 5 min at 2000×g and the supernatant used for IgM detection. Four consecutive dilutions of the serum, e.g., 1:20, 1:80, 1:320 and 1:1280, were prepared in buffer (PBS with 2 % BSA) and 20 µL of each dilution or control were added into the wells of a thawed slide. The slide was incubated for 1 h at 37 °C in a humidity chamber, washed with PBS and allowed to dry. The secondary antibody (goat anti-human IgM (H+L)/FITC and anti-IgG (Fc-γ specific)/FITC; Invitrogen) was diluted 1:50 in PBS with 2 % BSA. Subsequently, 10 µL of the secondary antibody dilution was mixed with 10 µL of 0.1 % Evans Blue (Sigma) in water and added per well. After 1 h of incubation at 37 °C the slide was washed with PBS, dried and covered with mounting medium (e.g., ROTI®Mount FluorCare from Carl Roth). Evaluation of the staining was done by fluorescence microscopy.