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.