TBEV antibody detection in rodents
Serum samples were screened for the presence of TBEV antibodies, using an in-house indirect immunofluorescence assay developed to capture specific IgG antibodies. ELISA plates were coated (2µg /well) with a purified SNAP-TBEV EDIII recombinant protein or a non-relevant SNAP. The recombinant SNAP-TBEV EDIII protein was produced on S2 cell lines from the synthetic gene encoding the soluble ecto-domain (residues E293 to E399) from TBEV strain Kumlinge A52 cloned into a pMT/BiP/V5-HisA vector (Life Technologies, France) according to the manufacturer’s recommendations (Beck et al., 2015). For the non-relevant SNAP, we chose a production with a pMT/BiP/V5-HisA vector alone without an added coding gene. After overnight coating with antigen the plates were saturated with 1% BSA in PBS solution. Assays were performed in duplicate with 100µl of serum samples (diluted 1:500 in PBS), tested on SNAP-TBEV EDIII recombinant protein and on the SNAP production on the same plate. Following incubation for 1 hour at 37°C the plates were washed three times in PBS-Tween 0.05%. The second antibody was a polyclonal goat anti-mouse conjugated with horseradish peroxidase (Dako), diluted to 1:500. After 1 hour at 37°C the wells were washed four times with 300μl of washing buffer. The bound peroxidase was revealed with 100µl 1.8mM of o-Phenylenediamine (Invitrogen) in PBS and 0.02% H2O2 according to the manufacturer’s instructions. After 40min in the dark at room temperature, the enzyme reaction was stopped with 0.5M H2SO4, and the optical density (OD) was measured at 492nm on an automatic plate reader.
The positive controls used to validate the ELISA plate protocol consisted of serum from two groups of three Swiss mice that had previously been immunised twice with the recombinant SNAP-TBEV EDIII protein and used at a dilution of 1:5000. The negative controls were three Swiss mice that had not been immunised twice with the recombinant SNAP-TBEV EDIII protein. The samples were considered positive when the duplicate mean OD values read for SNAP-TBEV EDIII minus the mean OD values read for SNAP was higher than 0.100 OD.